Inositol phosphate compounds for use in increasing tissular perfusion

ABSTRACT

The present invention relates to inositol phosphates, analogs, derivatives and pharmaceutically acceptable salts thereof, for use in increasing tissular perfusion and/or oxygenation in a subject in need thereof, in particular peripheral arterial disease. The present invention also relates to pharmaceutical compositions comprising said inositol phosphates, analogs, derivatives and pharmaceutically acceptable salts thereof, and their use in increasing tissular perfusion and/or oxygenation and for treating and preventing peripheral arterial disease.

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/ES2020/070070, filed on Jan. 30, 2020, which claims the prioritybenefits of European Application No. EP19382061.0, filed on Jan. 30,2019, and U.S. Provisional Application No. 62/913,259, filed on Oct. 10,2019, all of which are herein incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to the use of inositol phosphates (IP),their analogs and derivatives for increasing tissular perfusion and/oroxygenation. The present invention also relates to pharmaceuticalcompositions comprising said IP, their analogs and derivatives and theiruse in animal and human health.

BACKGROUND ART

Peripheral arterial disease (PAD) is a common disorder characterized bystenosis and/or obstruction of the lower limb arteries leading to adecreased muscle perfusion and oxygenation. PAD represents a majorpublic health issue and poses a high risk of long-term suffering. PADincreases the risk of tissue death (gangrene), amputation and prematuredeath.

PAD is the result of ischemia in the lower limbs. Its principal cause isatherosclerosis. In its mild form, PAD may be limited to intermittentclaudication and pain in the lower extremities. Lower extremity PAD is amajor cause of disability and mobility loss in older men and women andhas a decisive impact on quality of life.

The prevalence of PAD is ˜12% in the adult population. This prevalenceincreases to over 20% in the population group with more than 70 years ofage. PAD now affects more than eight million men and women in the UnitedStates only. It is estimated that more than 200 million individualssuffer PAD worldwide. The prevalence of PAD is likely to increase in thenear future as the general population grows older and the incidence ofobesity-related type 2 diabetes increases. Cigarette smoking is alsoanother significant risk factor. PAD patients have increased rates ofcardiovascular (CV) morbidity and mortality, faster rates of functionaldecline, and increased rates of mobility loss compared to the generalpopulation.

The protocol objectives for the treatment of PAD patients includereducing CV event rates, improving functional performance, andpreventing functional decline and the loss of mobility. Restoring orimproving blood perfusion to the limbs can help to achieve these goals.

While endovascular and lower extremity revascularization proceduressignificantly improve walking performance in PAD patients,revascularization procedures are not therapeutic options for many ofthem, either due to the presence of comorbid diseases or because thelocation and type of atherosclerotic disease in the lower extremities isnot amenable to revascularization. Revascularization is invasive,costly, and associated with risks, especially for older patients. Forthese reasons, there is a demand from clinicians for medical therapiesthat improve lower extremity functioning in PAD patients which areeffective, accessible and well tolerated.

At the present, only two medicaments have been approved by the FederalDrug Administration (FDA) for improving walking performance in peoplewith PAD: pentoxifylline (1984) and cilostazol (1999). No new drugs havebeen approved for treating intermittent claudication since then.Furthermore, in recent studies in PAD patients, pentoxifylline has notsignificantly improved intermittent claudication symptoms or maximalwalking distance more than placebo. Recently published clinical practiceguidelines recommend against prescription of pentoxifylline forintermittent claudication symptoms, due to a lack of therapeuticbenefit.

Cilostazol is a phosphodiesterase inhibitor that provides approximately25% to 40% improvement in treadmill walking performance in people withsymptomatic PAD. Cilostazol is a phosphodiesterase type 3 inhibitor thatacts by increasing the intracellular concentration of cyclic adenosinemonophosphate; in the process, the drug suppresses platelet aggregationand serves as a direct arterial vasodilator, improving of bloodperfusion. However, the mechanism by which cilostazol improves walkingability in PAD patients remains unclear.

Side effects of cilostazol include headache, diarrhea, palpitations, andlightheadedness. There is a black box warning against prescribingcilostazol to subjects with history of cardiovascular diseases.Cilostazol should not be administered to PAD patients who also haveheart failure. Cilostazol interacts with drugs prescribed regularly topatients with renal impairment or cardiovascular diseases, such ascinacalcet, clopidogrel and ibandronate, thus increasing the risk of anadverse reaction to these patients arising from the combined usecilostazol with other drugs.

In conclusion, medical therapies for symptomatic relief are limited,surgical or endovascular interventions are useful for some individuals,but long-term results are often disappointing. As a result, there is aneed for developing more effective and safer new therapies for treatingPAD.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a compound ofgeneral formula I, or a pharmaceutically acceptable salt thereof, foruse in increasing tissular perfusion and/or oxygenation in a subject inneed thereof.

where R₁, R₃, R₅, R₇, R₉ and R₁₁ are independently selected from OH, aradical of formula II, III, IV and a heterologous moiety:

with the condition that:at least one of R₁, R₃, R₅, R₇, R₉ and R₁₁ is selected from a radical offormula II, III and IV, andzero, one, two or three of R₁, R₃, R₅, R₇, R₉ and R₁₁ is a heterologousmoiety.

In another aspect, the present invention relates to a compound ofgeneral formula I, as defined above, for use in the treatment orprevention of ischemia in a subject in need thereof. In a version ofthis aspect, the invention refers to a compound of general formula I, asdescribed above, for use in the treatment or prevention of anischemia-related disease or condition in a subject in need thereof.

In some aspects, the present invention refers to a compound of generalformula I, as defined above, wherein the heterologous moiety is selectedfrom a radical of formula V, a radical of formula VI and a radical offormula VII:

and wherein n is an integer in the range from 2 to 200, and R₁₃ isselected from H, methyl or ethyl.

In a further aspect, the invention also relates to a method forincreasing tissular perfusion and/or oxygenation which comprisesadministering a therapeutically effective amount of a compound offormula I, as defined above, together with pharmaceutically acceptableexcipients or carriers, to a subject in need thereof. This aspect mayalso be formulated as the use of a compound of formula I, as definedabove, for the manufacture of a medicament for increasing tissularperfusion and/or oxygenation in a subject in need thereof.

In another aspect, the invention also relates to a method for treatingor preventing ischemia and/or an ischemia-related disease or conditionwhich comprises administering a therapeutically effective amount of acompound of formula I, as defined above, together with pharmaceuticallyacceptable excipients or carriers, to a subject in need thereof. Thisaspect may also be formulated as the use of a compound of formula I, asdefined above, for the manufacture of a medicament for treating orpreventing ischemia and/or an ischemia-related disease or condition in asubject in need thereof.

In a further aspect, the invention relates to a method for treating orpreventing peripheral arterial disease which comprises administering atherapeutically effective amount of a compound of formula I, as definedabove, together with pharmaceutically acceptable excipients or carriers,to a subject in need thereof. This aspect may also be formulated as theuse of a compound of formula I, as defined above, for the manufacture ofa medicament for treating or preventing peripheral arterial disease in asubject in need thereof.

The compounds of the present invention are particularly useful forincreasing tissular perfusion and/or oxygenation in the lower limbs and,especially, for the treatment or prevention of peripheral artery disease(PAD) and closely related conditions such as critical limb ischemia(CLI). These compounds also exhibit many advantageous properties (e.g.,better safety profile) in comparison to cilostazol, the reference drugcurrently indicated for the treatment of PAD.

The invention also provides a pharmaceutical composition comprising atleast one compound of formula I, as defined above, for use in: (i)increasing tissular perfusion and/or oxygenation, (ii) treating orpreventing ischemia and/or an ischemia-related disease, and/or (iii)treating or preventing PAD in a subject in need thereof. This aspect mayalso be formulated as the use of a pharmaceutical composition comprisingat least one compound of formula I, as defined above, for themanufacture of a medicament for: (i) increasing tissular perfusionand/or oxygenation, (ii) treating or preventing ischemia and/or anischemia-related disease, and/or (iii) treating or preventing PAD in asubject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows representative examples of inositol phosphate analogs inwhich two out of six X are OPSO₂ ²⁻ and the remaining X are OSO₃. Twospecific forms of4,6-di-(O-thiophosphate)-inositol-1,2,3,5-tetra-O-sulfate are shown.

FIG. 2 shows inositol phosphate analogs and inositol phosphatederivatives that can be used to practice the methods of the presentinvention. The molecules shown aremyo-inositol-pentakisphosphate-2-PEG400, myo-inositol hexakissulfate(myo-inositol hexasulfate), and scyllo-myo-inositol hexakissulfate(scyllo-inositol hexasulfate)

FIG. 3 shows inositol phosphate analogs and inositol phosphatederivatives that can be used to practice the methods of the presentinvention. X represent independently phosphorus and/or sulfur containinggroups (e.g., phosphate, sulfate, or thiophosphate). R¹ represents aheterologous moiety (e.g., PEG or PG).

FIG. 4 shows exemplary inositol phosphate analogs and inositol phosphatederivatives that can be used to practice the methods of the presentinvention. R¹ represents a heterologous moiety (e.g., PEG or PG). n canbe between 2 and 200.

FIG. 5 shows exemplary inositol phosphate analogs and inositol phosphatederivatives that can be used to practice the methods of the presentinvention. n can be between 2 and 200.

FIG. 6 shows exemplary inositol phosphate analogs and inositol phosphatederivatives that can be used to practice the methods of the presentinvention. n can be between 2 and 200.

FIG. 7 shows blood flow in posterior limbs in a rat model at D0 measuredby doppler laser imaging. Blood flow is shown in normalized perfusionunits (PU). Normalization is obtained by comparing the raw data to thegroup 1 data at D0.

FIG. 8 shows blood flow in posterior limbs in a rat model at D6 measuredby doppler laser imaging. Blood flow is shown in normalized perfusionunits (PU). Normalization is obtained by comparing the raw data to thegroup 1 data at D6.

FIG. 9 shows blood flow in posterior limbs in a rat model at D12measured by doppler laser imaging. Blood flow is shown in normalizedperfusion units (PU). Normalization is obtained by comparing the rawdata to the group 1 data at D12.

FIG. 10 shows inhibition percentage of aorta calcification in a VitD ratmodel at D12. Calcium level at sacrifice time was measured by ICP-OES.

FIG. 11 shows blood flow in posterior limbs in a rat model at D12 andD18 (6 days after the interruption of treatment) measured by dopplerlaser imaging. Blood flow is shown in normalized perfusion units (PU).Normalization is obtained by comparing the raw data to the group 1 dataat D12 and D18.

FIG. 12 shows (A) Maximum Walking Distance (MWD) and (B) Maximum WalkingTime (MWT) in a rat model at D10 measured by treadmill test. MaximumWalking Distance is shown in meters (m) and Maximum Walking Time inminutes (min).

FIG. 13 shows Maximum Walking Distance (MWD) in a rat model at D17 (5days after the interruption of treatment) measured by treadmill test.Maximal Walking Distance is shown in meters (m) up to 40 min of walkingtime.

FIG. 14 shows inhibition percentage of aorta calcification in a VitD ratmodel at D24 (12 days after the interruption of treatment). Calciumlevel at sacrifice time was measured by ICP-OES.

FIG. 15 shows blood flow in posterior limbs in a rat model at D0, D5,and D13 (8 days after starting treatment) measured by doppler laserimaging. Blood flow is shown in normalized perfusion units (PU).Normalization is obtained by comparing the raw data to the group 1 dataat D0, D5, and D13.

FIG. 16 shows (A) Maximum Walking Distance (MWD) and (B) Maximum WalkingTime (MWT) in a rat model at D11 (7 days after starting treatment)measured by treadmill test. Maximum Walking Distance is shown in meters(m) and Maximum Walking Time in minutes (min).

FIG. 17 shows inhibition percentage of femoral arteries calcification ina VitD rat model at D13 (9 days after starting treatment). Calcium levelat sacrifice time was measured by ICP-OES.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds, pharmaceutical compositions,methods and routes of administration for use in increasing tissularperfusion and/or oxygenation. The invention also provides compounds,pharmaceutical compositions, methods and routes of administration foruse in the treatment or prevention of ischemia and ischemia-relateddiseases and conditions.

The compounds of the present invention are particularly useful forincreasing tissular perfusion and/or oxygenation in the lower limbs and,especially, for the treatment or prevention of peripheral artery disease(PAD) and related conditions such as critical limb ischemia (CLI). Thesecompounds also exhibit many advantageous properties in comparison toother approved drugs for the treatment of PAD and CLI.

1. Definitions of General Terms and Expressions

The present invention includes embodiments in which exactly one memberof the group is present in, employed in, or otherwise relevant to agiven product or process. It also includes embodiments in which morethan one, or all of the group members are present in, employed in, orotherwise relevant to a given product or process.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by a skilled person in theart to which this description is related. For example, the Pei-Show J,Concise Dictionary of Biomedicine and Molecular Biology, 2nd Ed. (CRCPress, Boca Raton, Fla., USA 2002); Lackie J, The Dictionary of Cell andMolecular Biology, 5^(th) Ed. (Academic Press, Cambridge, Mass., USA2013) and Cammack R, et al., Oxford Dictionary of Biochemistry andMolecular Biology, 2^(nd) Ed., (Oxford University Press, Oxford, G B,2006) provide the skilled person with a general dictionary of many ofthe terms used in this description.

Units, prefixes, and symbols are denoted in their Systeme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Where a range of values is recited, it is tobe understood that each intervening integer value, and each fractionthereof, between the recited upper and lower limits of that range isalso specifically disclosed, along with each subrange between suchvalues. The upper and lower limits of any range can independently beincluded in or excluded from the range, and each range where either,neither or both limits are included is also encompassed within theinvention.

Where a value is explicitly recited, it is to be understood that valueswhich are about the same quantity or amount as the recited value arealso within the scope of the invention. Where a combination isdisclosed, each subcombination of the elements of that combination isalso specifically disclosed and is within the scope of the invention.Conversely, where different elements or groups of elements areindividually disclosed, combinations thereof are also disclosed. Whereany element of an invention is disclosed as having a plurality ofalternatives, examples of that invention in which each alternative isexcluded singly or in any combination with the other alternatives arealso hereby disclosed; more than one element of an invention can havesuch exclusions, and all combinations of elements having such exclusionsare hereby disclosed.

The term “and/or” as used herein is to be taken as the specificinvention of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B”, “A or B”, “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

The term “around” as used herein and as applied to one or more values ofinterest, refers to a value that is similar to a stated reference value.In certain aspects, the term “around” refers to a range of values thatfall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

The terms “critical limb ischemia” or “CLI” as used herein refer to asevere obstruction of the arteries which markedly reduces blood flow tothe extremities and progresses to the point of severe pain and even skinulcers, sores, or gangrene. Critical limb ischemia is a very severecondition of peripheral artery disease. In some aspects, theadministration of the inositol phosphate of the present invention (e.g.,myo-inositol hexaphosphate) to a subject in need thereof improves itscapacity for walking faster and for longer distances in comparison whenuntreated.

The term “compound” as used herein is meant to include all isomers andisotopes of the structure depicted. As used herein, the term “isomer”means any geometric isomer, tautomer, zwitterion, stereoisomer,enantiomer, or diastereomer of a compound. Compounds can include one ormore chiral centers and/or double bonds and can thus exist asstereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers)or diastereomers (e.g., enantiomers (i.e., (+) or (−)) or cis/transisomers). The present invention encompasses any and all isomers of thecompounds described herein, including stereomerically pure forms (e.g.,geometrically pure, enantiomerically pure, or diastereomerically pure)and enantiomeric and stereoisomeric mixtures, e.g., racemates.Enantiomeric and stereomeric mixtures of compounds and means ofresolving them into their component enantiomers or stereoisomers arewell-known. A compound, salt, or complex of the present invention can beprepared in combination with solvent or water molecules to form solvatesand hydrates by routine methods.

The term “cilostazol” as used herein, refers to6-[4-(1-cyclohexyl-1H-tetrazol-5yl)butoxy]-3,4-dihydro-2(1H)-quinolinone[CAS-73963-72-1], a quinolinone derivative that inhibits cellularphosphodiesterase. The molecular formula and weight of cilostazol areC₂₀H₂₇N₅O₂ and 369.46 g/mol, respectively. Its structural formula is:

The term “effective amount” as used herein, and the related terms“effective dose” and “effective dosage” in reference to (i) a compoundof a general formula I (e.g. an inositol phosphate, an inositolphosphate analog, an inositol phosphate derivative, or a combinationthereof), or (ii) a pharmaceutical composition comprising at least oneof the item (i) compounds, is that amount sufficient to effectbeneficial or desired results. In some embodiments, the beneficial ordesired results are, for example, clinical results, and, as such, an“effective amount” depends upon the context in which it is beingapplied. In the context of administering a therapeutic agent thatincreases tissular perfusion and/or oxygenation, an effective amount ofa therapeutic agent is, for example, an amount sufficient for (a)augmenting tissular perfusion in a specific area, (b) stopping,reducing, slowing the progression or reverting ischemia in a specificarea or (c) improving the mobility or walking ability (e.g. velocity,distance) in a subject, as compared to the same parameters observed inthe subject before the administration of the therapeutic agent, or in apopulation of control subjects without administration of the therapeuticagent.

The term “ischemia” as used herein refers to a restriction in bloodsupply to tissues, causing a shortage of oxygen that is required formaintaining cellular metabolism. Ischemia comprises not onlyinsufficiency of oxygen, but also reduced availability of nutrients andinadequate removal of metabolic wastes. Ischemia can be partial (poorperfusion) or total.

The terms “maximal walking distance” or “MWD” as used herein refer tothe distance at which a subject could not continue to walk unassisteddue to exhaustion or extreme pain. In the context of assessing anincrease in MWD, said increment is evaluated by comparing a subject'sMWD values before and after treatment with a therapeutic agent, or bycomparing the subject's MWD values after treatment with a population ofcontrol subjects untreated with the therapeutic agent.

The terms “maximal walking time” or “MWT” as used herein refer to thetime at which a subject could not continue to walk unassisted due toexhaustion or extreme pain. In the context of assessing an increase inMWT, said increment is evaluated by comparing a subject's MWT valuesbefore and after treatment with a therapeutic agent, or by comparing thesubject's MWT values after treatment with a population of controlsubjects untreated with the therapeutic agent.

The terms “parenteral administration” and “administered parenterally” asused herein means modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid,intraspinal, epidural and, intrasternal injection and infusion (e.g.,kidney dialytic infusion).

The terms “peripheral arterial disease” or “PAD” as used herein refer toa narrowing of the peripheral arteries to the legs (most commonly),stomach, arms, and head. Symptoms include intermittent claudication(e.g., leg pain when walking which resolves with rest), skin ulcers,bluish skin, cold skin, or poor nail and hair growth.

The terms “prevent”, “preventing” and “prevention” as used herein referto inhibiting the inception or decreasing the occurrence of a disease orcondition in a subject (e.g., avoiding the development of ischemictissue in the limbs).

The term “SNF472” as used herein refers to an intravenous myo-inositolhexaphosphate hexasodium formulation. SNF472 is manufactured bydissolving myo-inositol hexaphosphate hexasodium in saline solution,followed by pH adjustment and aseptic filtration. SNF472 is prepared atthree different strengths: (a) (i) 20 mg/mL and (ii) 90 mg/mL in 5 mLsingle-use vials, formulated in saline solution, pH 5.8 to 6.2 and (b)30 mg/L in 10 mL single-use vials, formulated in saline solution, pH 5.6to 6.4.

The terms “subject”, “individual”, “animal” or “mammal” as used hereinis meant any subject, particularly a mammalian subject, for whomdiagnosis, prognosis, or therapy is desired. Mammalian subjects include,but are not limited to, humans, domestic animals, farm animals, zooanimals, sport animals, pet animals such as dogs, cats, guinea pigs,rabbits, rats, mice, horses, cattle, cows; primates such as apes,monkeys, orangutans, and chimpanzees; canids such as dogs and wolves;felids such as cats, lions, and tigers; equids such as horses, donkeys,and zebras; bears, food animals such as cows, pigs, and sheep; ungulatessuch as deer and giraffes; rodents such as mice, rats, hamsters andguinea pigs; and so on. In certain aspects, the subject is a humansubject. In some aspects, the subject is a human patient with a reducedtissular perfusion and/or oxygenation in the lower limb muscles or atrisk of developing said condition. In some further aspects, the subjectis a human patient with ischemia and/or an ischemia-related disease orcondition, or at risk of developing said ischemia, ischemia-relateddisease or condition.

The term “substantially” as used herein refers to the qualitativecondition of exhibiting total or near-total extent or degree of acharacteristic or property of interest. A person skilled in thebiological arts will understand that biological and chemical phenomenararely, if ever, go to completion and/or proceed to completeness orachieve or avoid an absolute result. The term “substantially” istherefore used herein to capture the potential lack of completenessinherent in many biological and chemical phenomena.

The term “tissular perfusion” as used herein refers to the flow of bloodor other perfusate through the vessels of a specific tissue or organ.“Increase tissular perfusion” or “increasing tissular perfusion” as usedherein relate to an increment in the blood flow in a specific tissuearea in a subject after administering an inositol phosphate of thepresent invention as compared to the same parameters observed in thesubject before the administration of said therapeutic agent, or in apopulation of control subjects without administration of saidtherapeutic agent.

The terms “treat” or “treatment” as used herein refer to theadministration of compound or pharmaceutical composition of the presentinvention for (i) slowing, (ii) inhibiting the progression, (iii)stopping, or (iv) reverting the progression of a disease or conditionafter its clinical signs have appeared. Control of the diseaseprogression is understood to mean the beneficial or desired clinicalresults that include, but are not limited to, reduction of the symptoms,reduction of the duration of the disease, stabilization of pathologicalstates (specifically to avoid additional deterioration), delaying theprogression of the disease, improving the pathological state andremission (both partial and total). The control of progression of thedisease also involves an extension of survival compared with theexpected survival if treatment was not applied. Within the context ofthe present invention, the terms “treat” and “treatment” referspecifically to (a) increasing tissular perfusion and/or oxygenation or(b) stopping, reducing, slowing the progression or reverting thedevelopment of ischemic tissue, especially in the lower limbs, or (c)improving the mobility or walking ability (e.g., velocity, distance,endurance) in a subject administered with the compounds or thepharmaceutical compositions of the present invention.

The term “walking ability” as used herein refers to the capacity of asubject for mobilizing autonomously without assistance. The parametersMWD and MWT are indicative of a subject's walking ability.

2. Compounds

The compounds for use in the present invention are inositol phosphates,as defined in the first aspect of the invention, as well as analogs andderivatives thereof. The term “inositol phosphate” as used herein refersto a compound with an inositol ring and one, two, three, four, five, orsix phosphate groups, or a combination thereof. Myo-inositolhexaphosphate (IP6) is an exemplary inositol phosphate of the presentinvention. In some aspects, the inositol phosphate is pure (e.g., over99% of the inositol phosphate species are the same species, for example,IP6) or substantially pure (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the inositol phosphate species are the samespecies, for example, IP6). In some aspects, the inositol phosphate is amixture, e.g., comprising variable amounts of IP1, IP2, IP3, IP4, IP5,and IP6. In some aspects, the inositol phosphate is a racemic mixture.

The invention also contemplates inositol phosphate analogs. “Inositolphosphate analog” as used herein refers to a compound that has a ringwith different number of carbons with respect to an inositol ring (i.e.,5 or 7 carbons), and/or has at least one sulfate or thiophosphate group.For example, a compound comprising a ring with 5, 6, or 7 carbons and atleast one phosphate, sulfate, or thiophosphate group would be consideredan inositol phosphate analog.

The term “inositol phosphate derivative” as used herein refers to aninositol phosphate or inositol phosphate analog which contains aheterologous moiety (i.e., a group that is not a phosphate, a sulfate,or a thiophosphate). For example, an inositol pentasulfate comprising apolyethylene glycol heterologous moiety, or a myo-inositol hexaphosphatecomprising a polyglycerol heterologous moiety would be consideredinositol phosphate derivatives.

The term “heterologous moiety” as used herein refers to a radical in thecompound of formula I which is not a phosphate, a sulfate, or athiophosphate, and confers a desirable property to such compound. Forexample, a heterologous moiety (e.g., a polyglycerol or apolyethyleneglycol) can increase the solubility of the compound. In someaspects, a heterologous moiety can confer multiple desirable properties(e.g., polyglycerol and polyethyleneglycol can both increase thesolubility of a compound and reduce the clearance rate of the compound).

The terms “inositol phosphate of the invention” and “inositol phosphateof the present invention” as used herein is a generic term encompassing“inositol phosphate”, “inositol phosphate analog”, “inositol phosphatederivative” and combinations thereof. In some aspects, the term“inositol phosphate of the present invention” encompasses compositionscomprising an “inositol phosphate” an “inositol phosphate analog” an“inositol phosphate derivative” or a combination thereof, and at leastone additional therapeutic agent. In some aspects, the additionaltherapeutic agent comprises cilostazol, pentoxifylline or a combinationthereof.

Compounds of the present invention comprising a ring with 5, 6, or 7carbons and at least one sulfate, or thiophosphate group but without aphosphate group would still be considered an “inositol phosphate analog”or an “inositol phosphate analog” in the context of the presentinvention. Thus, the term “inositol phosphate of the present invention”encompasses not only phosphate-containing compounds but also compoundswithout phosphate groups that comprise a ring with 5, 6, or 7 carbonsand at least one sulfate, or thiophosphate group.

Representative inositol phosphates of the present invention arepresented in FIGS. 1-6. FIG. 3 present numerous examples of inositolphosphates, all of them in the myo conformation. Besides myo-inositol,the other naturally occurring stereoisomers of inositol are scyllo-,muco-, 1D-chiro-, 1L-chiro-, neo-inositol, allo-, epi-, andcis-inositol. As their names denote, 1L- and 1D-chiro inositol are theonly pair of inositol enantiomers, but they are enantiomers of eachother, not of myo-inositol. It is to be understood that any exemplaryinositol phosphate presented in the disclosure is not limited to therepresentative conformation displayed. Thus, for example, the examplespresented in FIG. 3 would also encompass the corresponding equivalentsin scyllo-, muco-, 1D-chiro-, 1L-chiro-, neo-inositol, allo-, epi-, andcis-inositol conformations. In its most stable conformation, themyo-inositol isomer assumes the chair conformation, which moves themaximum number of hydroxyls to the equatorial position, where they arefarthest apart from each other. In this conformation, the natural myoisomer has a structure in which five of the six hydroxyls (the first,third, fourth, fifth, and sixth) are equatorial, whereas the secondhydroxyl group is axial.

2.1. Inositol Phosphates, Analogs and Derivatives

In some aspects, at least one of R₁, R₃, R₅, R₇, R₉ and R₁₁ of thecompound of general formula I independently represents H, —X, —OX, —NHX,—NX₂, —SX, —OSO₃HX, —OSO₃X₂ or a compound of formula II, formula III orformula IV, where each X independently represents H, C₁₋₃₀ alkyl, C₂₋₃₀alkynyl or Cy₁, where C₁₋₃₀ alkyl, C₂₋₃₀ alkenyl and C₂₋₃₀ alkynyl areindependently optionally substituted with one or more R₁₄ and where Cy₁is optionally substituted by one or more R₁₅; Cy₁ represents acarbocyclic or heterocyclic three- to 10-membered ring, which can besaturated, partially unsaturated or aromatic, where said heterocycle hasbetween one and four heteroatoms selected from amongst O, S and N, wheresaid ring can be bound to the rest of the molecule via any available Catom and where Cy₁ is optionally fused to between one and four five- orsix-membered rings, each saturated, partially unsaturated or aromatic,carbocyclic or heterocyclic, and where said fused heterocycle cancontain one or two heteroatoms selected from amongst O, N and S; eachR₁₃ independently represents H, C₁₋₃₀ alkyl, —NH₂, —NHC₁₋₃₀ alkyl orN(C₁₋₃₀ alkyl)₂, where each C₁₋₃₀ alkyl is independently optionallysubstituted with one or more halogen, —OH, —CN and —NO₂ groups; and eachR₁₄ and R₁₅ independently represents OH, C₁₋₃₀ alkoxy, C₁₋₃₀alkyithionyl, C₁₋₃₀ acyloxy, phosphate, halogen, trihalo C₁₋₃₀ alkyl,nitrile azide.

In some ulterior aspects, each X independently represents H, C₁₋₃₀ alkylor Cy₁, where C₁₋₃₀ alkyl is optionally substituted by one or more R₁₄and where Cy₁ is optionally substituted by one or more R₁₅; and each R₁₄and R₁₅ independently represents —OH, C₁₋₃₀ alkoxy, C₁₋₃₀ alkyithionyl,C₁₋₃₀ acyloxy, phosphate, halogen, trihaloC₁₋₃₀alkyl, nitrile or azide.In some aspects, each X represents H, C₁₋₃₀alkyl or Cy₁. In someaspects, each X represents H.

In some additional aspects, at least one of radicals R₁, R₃, R₅, R₇, R₉and R₁₁ independently represents a compound of formula II, formula IIIor formula IV, each R₁₃ independently represents H, C₁₋₃₀ alkyl, —NH₂,—NHC₁₋₃₀ alkyl or —N(C₁₋₃₀ alkyl)₂, where each C₁₋₃₀ alkyl isindependently optionally substituted by one or more halogen, —OH, —CNand —NO₂ groups; and R₂, R₄, R₆, R₅, R₁₀ and R₁₂ independently representH.

In a further aspect, R₁, R₃, R₅, R₇, R₉ and R₁₁ independently representa compound of formula II, formula III, or formula IV, each R₁₃independently represents H or C₁₋₃₀ alkyl, where each C₁₋₃₀ alkyl isindependently optionally substituted by one or more halogen, —OH, —CNand —NO₂ groups; and R₂, R₄, R₆, R₅, R₁₀ and R₁₂ independently representH.

In an additional aspect, at least one of R₁, R₃, R₅, R₇, R₉ and R₁₁represent a compound of formula II, formula III, or formula IV, and eachR₁₃ independently represents H or C₁₋₃₀ alkyl. In another aspect, atleast one of R₁, R₃, R₅, R₇, R₉ and R₁₁ represent a compound of formulaII, formula III or formula IV, and each R₁₃ represents H.

In a particular aspect, the compound is inositol hexaphosphate (IP6). Inother aspects, the compound is inositol monophosphate (IP1), inositoldiphosphate (IP2), inositol triphosphate (IP3), inositol tetraphosphate(IP4), or inositol pentaphosphate (IP5). In some aspects, the compoundcomprises a combination of IP1, IP2, IP3, IP4, IP6 and IP6. In someaspects, the IP6 can form other inositol phosphates (IP5, IP4, IP3, IP2,IP1) by dephosphorylation in vivo. Inositol is assumed to mean anyisomeric form of the molecule, for example, myoinositol.

In some aspects, the compounds for use in the present invention arethose of formula I wherein:

R₇ is OSO₃ ⁻, and R₉, R₅, R₃, R₁ and R₁ are independently selected fromOPO₃ ²⁻, OPSO₂ ²⁻ or OSO₃ ⁻;R₉, R₅ and R₁ are OPO₃ ²⁻ and R₇, R₃ and R₁ are OSO₃ ⁻;R₉, R₅ and R₁ are OSO₃ ⁻ and R₇, R₃ and R₁ are OPO₃ ²⁻;R₃, R₁ and R₁ are OSO₃ ⁻ and R₉, R₇ and R₅ are OPO₃ ²⁻;R₃, R₁ and R₁ are OPO₃ ²⁻ and R₉, R₇ and R₅ are OSO₃ ⁻;R₇ and R₁ are OPO₃ ²⁻ and R₉, R₅, R₃, and R₁ are OPO₃ ²⁻.R₇ and R₁ are OSO₃ ⁻ and R₉, R₅, R₃, and R₁ are OPO₃ ²⁻.R₇ and R₅ are OPO₃ ²⁻ and R₉, R₃, R₁, and R₁ are OSO₃ ⁻; or,R₇ and R₅ are OSO₃ ⁻ and R₉, R₃, R₁, and R₁ are OPO₃ ²⁻.

The inositol phosphates of the present invention also encompasscompounds that are produced as metabolites during physiologicaldephosphorylation (or desulfation or dethiosulfation in the case ofcompounds comprising sulfate or thiophosphate groups).

In some aspects, the compound administered in a dosage according to themethods disclosed herein is a prodrug that after undergoing hydrolysisor other intracellular or extracellular processing yields an inositolphosphate of the present invention.

The inositol phosphates of the present invention encompass also anycombination of the inositol phosphate, inositol phosphate analogs, andderivatives thereof disclosed herein.

All compounds of formula I contain radicals with C—O—P or C—O—S bonds,which provide the compounds with an affinity for calcium-containingcrystals and a sufficiently labile bond to be hydrolyzed in vivo,thereby preventing irreversible binding to calcium-containing crystalssuch as the hydroxyapatite (HAP) in bone, which would have a negativeimpact on bone remodeling, as is the case with bisphosphonates whenadministered long term as said compounds contain P—C—P bonds that cannotbe hydrolyzed by the body. At the other extreme are phosphorylatedcompounds that do not contain said C—O—P bonds, such as pyrophosphates,the P—O—P bonds of which mean that they are too readily hydrolyzed inthe intestine, thus meaning that only parenteral administration isfeasible. The compounds of the present invention, with C O—P bonds,C—O—S bonds, and combinations thereof represent an adequate midpoint dueto the efficacy thereof and the fact that the body presents mechanismsfor eliminating said compounds, thus reducing the risk of side effects(e.g., compounds with P—C—P bonds can present half-lives of severalmonths which in vivo, thereby affecting, e.g., bone remodeling).

The term “alkyl” or “alkyl group” in the context of the presentinvention refers to a saturated hydrocarbon moiety, which can be linear,branched, cyclic or cyclic with linear or branched side chains. The termalkyl includes partially unsaturated hydrocarbons such as propenyl.Examples are methyl, ethyl, n- or isobutyl, n- or cyclohexyl. The termalkyl can extend to alkyl groups linked or bridged by hetero atoms.Hetero atoms in the context of the present invention are nitrogen (N),sulfur (S) and oxygen (O).

An “amine function” or “amine group” is a function NR′R″, with R′ and R″selected independently from hydrogen and C₁-C₅ alkyl. In some aspects,R′ and R″ are selected from hydrogen and C₁-C₃ alkyl. A “hydroxyfunction” or “hydroxy group” is OH.

A “thiol function” or “thiol group” is SH. A “carboxylic acid function”or “carboxylic acid group” is COOH or its anion, COO—. A “carboxylicamide” is CONR′R″, with R′ and R″ independently having the meaningsindicated above. A “sulfonic acid” is SO₃H. A “sulfonic acid amide” isSO₂NR′R″, with R′ and R″ independently having the meanings indicatedabove.

A “C₁-C₃ alkyl” in the context of the present invention refers to asaturated linear or branched hydrocarbon having 1, 2, or 3 carbon atoms,wherein one carbon-carbon bond can be unsaturated and one CH₂ moiety canbe exchanged for oxygen (ether bridge). Non-limiting examples for aC₁-C₃ alkyl are methyl, ethyl, propyl, prop-2-enyl and prop-2-inyl.

A “C₁-C₅ alkyl” in the context of the present invention refers to asaturated linear or branched hydrocarbon having 1, 2, 3, 4 or 5 carbonatoms, wherein one or two carbon-carbon bond can be unsaturated and oneCH₂ moiety can be exchanged for oxygen (ether bridge). Non-limitingexamples for a C₁-C₅ alkyl include the examples given for C₁-C₃ alkylabove, and additionally n-butyl, 2-methylpropyl, tert-butyl,3-methylbut-2-enyl, 2-methylbut-3-enyl, 3-methylbut-3-enyl, n-pentyl,2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl,1,2-dimethylpropyl, but-3-enyl, but-3-inyl and pent-4-inyl. A “C₃ ⁻ C₁₀alkyl” in the context of the present invention refers to a saturatedlinear or branched hydrocarbon having 3, 4, 5, 6, 7, 8, 9 or 10 carbonatoms, wherein 1, 2 or 3 carbon-carbon bonds can be unsaturated and oneCH₂ moiety can be exchanged for oxygen (ether bridge).

The term “C₁₋₃₀ alkyl” as a group or part of a group, refers to a linearor branched chain alkyl group containing between 1 and 30 carbon atomsincluding, amongst others, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, decyl and dodecylgroups.

The term “C₂₋₃₀ alkenyl” refers to a linear or branched alkyl chaincontaining between 2 and 30 carbon atoms and also contains one or moredouble bonds. Examples include, amongst others, ethenyl, 1-propenyl,2-propenyl, isopropenyl 1-butenyl, 2-butenyl, 3-butenyl and1,3-butadienyl.

The term “C₂₋₃₀ alkynyl” refers to a linear or branched alkyl chaincontaining between 2 and 30 carbon atoms and also contains one or moretriple bonds. Examples include, amongst others, ethynyl, 1-propynyl,2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl and 1,3-butadiynyl.

A “Cy₁ group” refers to a three- to 10-membered carbocyclic orheterocyclic ring that can be saturated, partially unsaturated oraromatic and which is bound to the rest of the molecule via anyavailable C atom. When heterocyclic, Cy₁ contains between one and fourheteroatoms selected from amongst N, O and S. Moreover, Cy₁ canoptionally be fused with up to four five- or six-membered carbocyclic orheterocyclic rings, which can be saturated, partially unsaturated oraromatic. If the fused ring is a heterocycle, said ring contains one ortwo heteroatoms selected from amongst N, O and S. Examples of Cy₁include, amongst others, phenyl, naphthyl, thienyl, furyl, pyrrolyl,thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl,1,2,4-triazolyl, tetrazolyl, 1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl,pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, benzimidazolyl,benzofuranyl, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl,benzothiazolyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, azetidinyl and aziridinyl.

A “C₁₋₃₀ alkoxy group” as a group or part of a group refers to anOC₁₋₃₀alkyl group, where the C₁₋₃₀alkyl part has the same meaning asabove. Examples include methoxy, ethoxy, propoxy, isopropoxy, butoxy,isobutoxy, sec-butoxy and tert-butoxy.

A “C₁₋₃₀ alkylthionyl group” as a group or part of a group refers to anSOC₁₋₃₀alkyl group, where the C₁₋₃₀alkyl part has the same meaning asabove. Examples include methylthionyl, ethylthionyl, propyithionyl,isopropyithionyl, butylthionyl, isobutyithionyl, sec-butylthionyl andtert-butylthionyl.

A “C₁₋₃₀ acyloxy group” as a group or part of a group refers to aCOC₁₋₃₀alkyl group, where the C₁₋₃₀alkyl part has the same meaning asabove. Examples include acetyl, ethanoyl, propanoyl and2,2-diisopropylpentanoyl.

A “halogen radical” or the halo abbreviation thereof refers to fluorine,chlorine, bromine and iodine.

A “trihalo C₁₋₃₀ alkyl group” refers to a group resulting from thesubstitution of three hydrogen atoms of a C₁₋₃₀alkyl group by threehalogen radicals as defined above. Examples include, amongst others,trifluoromethyl, tribromomethyl, trichloromethyl, triiodomethyl,trifluoroethyl, tribromoethyl, trichloroethyl, triiodoethyl,tribromopropyl, trichloropropyl and triiodopropyl.

An “—NHC₁₋₃₀ alkyl group” refers to a group resulting from thesubstitution of one hydrogen atom of an —NH₂ group by a C₁₋₃₀ alkylgroup as defined above. Examples include, amongst others, methylamine,ethylamine, propylamine, butylamine and pentylamine.

A “—N(C₁₋₃₀ alkyl)₂ group” refers to a group resulting from thesubstitution of two hydrogen atoms of an —NH₂ group by a C₁₋₃₀ alkylgroup as defined above. Examples include, amongst others, dimethylamine,diethylamine, diisopropylamine, dibutylamine and diisobutylamine.

The expression “optionally substituted by one or more” signifies thepossibility that a group can be substituted by one or more (e.g., by 1,2, 3 or 4) substituents. In some aspects, a group can be substituted by1, 2 or 3 substituents and even by 1 or 2 substituents provided that thegroup has sufficient positions that can be substituted available. Ifpresent, the substituents can be the same or different and can belocated at any available position.

In some aspects, the inositol phosphates of the present inventioncomprise the compounds disclosed in WO2017098033 and WO2017098047, andUS U.S. Pat. No. 9,358,243. In some aspects, the inositol phosphates ofthe present invention used comprise the compounds disclosed in FIGS.1-6.

In some aspects, the inositol phosphates, inositol phosphate analogs,and derivatives thereof, comprise compounds of formula (VIII), formula(IX), or formula (X):

wherein each X independently is selected from OPO₃ ²⁻, OPSO₂ ²⁻, orOSO₃; Z is an alkyl chain comprising 1 to 3 carbon and/or hetero atoms,optionally comprising a group X, wherein X is also selected from OPO₃²⁻, OPSO₂ ²⁻, or OSO₃ ⁻; and, R¹ is an optional heterologous moiety (Seesection 2.2. below). In some aspects, the molecule comprises more thanone heterologous moiety, in which case the heterologous moieties can bethe same or be different.

In some aspects, Z, as used in formula (VIII), is CH₂, CHX, CHR¹, CXR¹,CH₂—CH₂, CH₂—CHX, CHX—CHX, CHR¹—CHX, CXR¹—CHX, CHR¹—CH₂, CXR¹CH₂,CHR¹—CHOH, CH₂—CH₂—CH₂, CH₂—O—CH₂, CHOH—CH₂—CH₂, CHOH—CHOH—CHR¹,CHOH—CHR¹—CHOH, CHX—CH₂—CH₂, CH₂—CHX—CH₂, CHX—CHX—CH₂, CHX—CH₂—CHX orCHX—CHR¹—CHX, wherein X independently is selected from OPO₃ ²⁻, OPSO₂²⁻, and OSO₃ ⁻.

In some aspects, Z, as used in formula (VIII), is (CHX)_(p)CHX(CHX)_(q);wherein p and q each independently from the other have a value from 0 to2, with the proviso that (p+q) has a value of 0, 1 or 2; one or two orthree X can be a heterologous moiety (e.g., PEG) and the remaining X areindependently selected from OPO₃ ²⁻, OPSO₂ ²⁻, and OSO₃ ⁻. In someaspects, not all X of Z are PO₃ ²⁻. In some aspects, not all X of Z areOSO₃ ⁻.

In some aspects, one, two, or three of the X in compounds of formula(VIII), formula (IX), or formula (X) can be heterologous moiety and theremaining X can independently be selected from OPO₃ ²⁻, OPSO₂ ²⁻, orOSO₃.

Formula (VII) above describes a five-membered, six-membered, orseven-membered alkyl ring, and the optional heterologous moiety ormoieties is/are attached to one of the carbon atoms forming the ring.

In some aspects, the inositol phosphates, inositol phosphate analogs,and derivatives thereof used, e.g., in the methods and compositionsdisclosed herein, comprise compounds of formula (XI) or formula (XII):

wherein:X² is OSO₃ ⁻, and X¹, X³, X⁴, X⁵ and X⁶ are independently selected fromOPO₃ ²⁻, OPSO₂ ²⁻ or OSO₃ ⁻;X¹, X³ and X⁵ are OPO₃ ²⁻ and X², X⁴ and X⁶ are OSO₃ ⁻;X¹, X³ and X⁵ are OSO₃ ⁻ and X², X⁴ and X⁶ are OPO₃ ²⁻;X⁴, X⁵ and X⁶ are OSO₃ ⁻ and X¹, X² and X³ are OPO₃ ²⁻;X⁴, X⁵ and X⁶ are OPO₃ ²⁻ and X¹, X² and X³ are OSO₃ ⁻;X² and X⁵ are OPO₃ ²⁻ and X¹, X³, X⁴, and X⁶ are OPO₃ ²⁻;X² and X⁵ are OSO₃ ⁻ and X¹, X³, X⁴, and X⁶ are OPO₃ ²⁻;X² and X³ are OPO₃ ²⁻ and X¹, X⁴, X⁵, and X⁶ are OSO₃ ⁻; or,X² and X³ are OSO₃ ⁻ and X¹, X⁴, X⁵, and X⁶ are OPO₃ ²⁻

In some aspects, the inositol phosphates of the present invention ormetabolites thereof can be detected and/or quantified using the methodsdisclosed in U.S. Pat. No. 9,612,250. See also, U.S. Pat. Nos.8,377,909, 8,778,912, and US20070066574.

The compounds disclosed herein can be present in any form commonly usedin pharmaceutical technology. Particular aspects include, but are notlimited to, the sodium salt, magnesium salt, potassium salt, ammoniumsalt, free acid, or a mixture of the preceding forms. Otherpharmaceutically acceptable salts are known to the skilled artisan andcan be readily obtained. In a particular aspect, the compound for use asdefined in the first aspect of the invention is a sodium salt, forexample, inositol hexaphosphate hexasodium.

The present invention also contemplates sodium salts of inositolmonophosphate, inositol diphosphate, inositol triphosphate, inositoltetraphosphate and inositol pentaphosphate in any of inositol isomericforms, in particular, myo-inositol. A particular example of thecompounds for use in the present invention is myo-inositol hexaphosphatehexasodium salt. Sodium salts provide several advantages in terms of themanufacturing and the level of impurities of the resulting IP6formulations.

2.2. Heterologous Moiety

In some aspects, the present invention refers to a compound of generalformula I, as defined above, wherein the heterologous moiety is selectedfrom a radical of formula V, a radical of formula VI and a radical offormula VII:

and wherein n is an integer in the range from 2 to 200, and R₁₃ isselected from H, methyl or ethyl.

In some aspects, compounds for use in the present invention, forexample, the inositol phosphate derivatives of the present invention,can comprise one or two radicals selected from the radicals of formulasV, VI and VII. These radicals are heterologous moieties conferring anadvantageous property with respect to a corresponding molecule lackingsuch heterologous moiety or moieties. Examples of said advantageousproperties that can be conferred by a heterologous moiety or acombination thereof to an inositol phosphate or inositol phosphateanalogs include, but are not limited to (a) an increase in solubility,(b) a decrease in degradation or metabolization rate, (c) an increase inplasma half-life, (d) a decrease in liver metabolization rate (e) adecrease in clearance rate, (f) a decrease of toxicity, (g) a decreaseof irritability and (h) reduced side effects among others. Theseadvantageous properties can be evaluated or quantified using methodsknown in the art without undue experimentation.

In some aspects, the heterologous moiety is, for instance, apolyethylene glycol (PEG) or a polyglycerol (PG). Thus, in certainaspects, the compound for use in the invention is any of the compoundsas defined in the aspects disclosed above comprising a heterologousmoiety, that is, one of the radicals of formula I is selected from theradicals of formulas V, VI and VII. In some aspects the heterologousmoiety comprises a polyethylene glycol (PEG). In certain aspects theheterologous moiety consists of polyethylene glycol, that is to say, atleast one of R₁, R₃, R₅, R₇, R₉ and R₁₁ of the compound of formula Iaccording to the first aspect of the invention is a radical of formulaV. Alternatively, the heterologous moiety comprises a polyglycerol. Incertain aspects the heterologous moiety consists on polyglycerol, thatis to say, at least one of R₁, R₃, R₅, R₇, R₉ and R₁₁ of the compound offormula I according to the first aspect of the invention is selectedfrom a radical of formula VI or VII. In other aspects the compound offormula I according to the first aspect of the invention contains one,two or three radicals selected from a radical of formula VI or VII, forexample two PEGs (radical of formula V), Three PEGs, two polyglycerols(radical of formula VI), three PGs, or any combinations thereof, forexample, one PEG and one PG, or two PEGs and one polyglycerol. Incertain aspects all of the remaining radicals of formula I (i.e., thosethat are not a radical selected from V, VI and VII) are a radicalselected from II, III and IV. In some aspects the compound of formula Iaccording to the first aspect of the invention contains two radicalsselected from a radical of formula VI or VII, for example two PEGs(radical of formula V) or two polyglycerols (radical of formula VI) orone PEG and one polyglycerol and the remaining radicals are all aradical of formula II. In some aspects R₃ and R₇ of the compound offormula I are selected from a radical of formula V, VI and VII. In someaspects, R₃ and R₇ of the compound of formula I are radicals of formulaV, and R₁, R₅, R₉ and R₁₁ of the compound of formula I are radicals offormula II.

The radicals of formulas V, VI and VII have R₁₃═H, methyl or ethyl and nis an integer from 2 to 200. In some aspects R₁₃═H. In particularaspects n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,177, 178, 179, 189, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,191, 192, 193, 194, 195, 196, 197, 198, 199, or 200.

In some aspects, n is between 2 and 10, between 10 and 20, between 20and 30, between 30 and 40, between 40 and 50, between 50 and 60, between60 and 70, between 70 and 80, between 80 and 90, between 90 and 100,between 100 and 110, between 110 and 120, between 120 and 130, between130 and 140, between 140 and 150, between 150 and 160, between 160 and170, between 170 and 180, between 180 and 190, or between 190 and 200.

In some specific aspects, n has a value from 2 to 200, from 2 to 20,from 10 to 30, or from 9 to 45.

In some aspects, the PEG is a branched PEG. Branched PEGs have three toten PEG chains emanating from a central core group.

In certain aspects, the PEG moiety is a monodisperse polyethyleneglycol. In the context of the present invention, a monodispersepolyethylene glycol (mdPEG) is a PEG that has a single, defined chainlength and molecular weight. mdPEGs are typically generated byseparation from the polymerization mixture by chromatography. In certainformulae, a monodisperse PEG moiety is assigned the abbreviation mdPEG.In some aspects, the PEG is a Star PEG. Star PEGs have 10 to 100 PEGchains emanating from a central core group.

In some aspects, the PEG is a Comb PEGs. Comb PEGs have multiple PEGchains normally grafted onto a polymer backbone.

In certain aspects, the PEG has a molar mass between 100 g/mol and 3000g/mol, particularly between 100 g/mol and 2500 g/mol, more particularlyof approx. 100 g/mol to 2000 g/mol. In certain aspects, the PEG has amolar mass between 200 g/mol and 3000 g/mol, particularly between 300g/mol and 2500 g/mol, more particularly of approx. 400 g/mol to 2000g/mol.

In some aspects, the PEG is PEG₁₀₀, PEG₂₀₀, PEG₃₀₀, PEG₄₀₀, PEG₅₀₀,PEG₆₀₀, PEG₇₀₀, PEG₅₀₀, PEG₉₀₀, PEG₁₀₀₀, PEG₁₁₀₀, PEG₁₂₀₀, PEG₁₃₀₀,PEG₁₄₀₀, PEG₁₅₀₀, PEG₁₆₀₀, PEG₁₇₀₀, PEG₁₅₀₀, PEG₁₉₀₀, PEG₂₀₀₀, PEG₂₁₀₀,PEG₂₂₀₀, PEG₂₃₀₀, PEG₂₄₀₀, PEG₂₅₀₀, PEG₁₆₀₀, PEG₁₇₀₀, PEG₁₈₀₀, PEG₁₉₀₀,PEG₂₀₀₀, PEG₂₁₀₀, PEG₂₂₀₀, PEG₂₃₀₀, PEG₂₄₀₀, PEG₂₅₀₀, PEG₂₆₀₀, PEG₂₇₀₀,PEG₂₅₀₀, PEG₂₉₀₀, or PEG₃₀₀₀. In one particular aspect, the PEG isPEG₄₀₀. In another particular aspect, the PEG is PEG₂₀₀₀.

In other particular aspects R₃ and/or R₇ of the compound of formula I isa radical of formula V where R₁₃ is H and n is an integer from 9 to 45.In other particular aspects R₃ and R₇ of the compound of formula I is aradical of formula V where R₁₃ is H and n is an integer from 9 to 45 andR₁, R₅, R₉ and R₁₁ are all a radical of formula II. In other particularaspects the compound of formula I is a sodium salt with R₃ andR₇=radical of formula V where R₁₃ is H and n is an integer from 9 to 45,and R₁, R₅, R₉ and R₁ are all a radical of formula II.

In some other aspects, the heterologous moiety is a polyglycerol (PG)described by the formula ((R₃—O—(CH₂—CHOH—CH₂O)_(n)—) with R₃ beinghydrogen, methyl or ethyl, and n having a value from 3 to 200. In someaspects, n has a value from 3 to 20. In some aspects, n has a value from10 to 30. In some alternatives of these aspects, n has a value from 9 to45. In some aspects, the heterologous moiety is a branched polyglyceroldescribed by the formula (R³—O—(CH₂—CHOR⁵—CH₂—O)_(n)—) with R⁵ beinghydrogen or a linear glycerol chain described by the formula(R³—O—(CH₂—CHOH—CH₂—O)_(n)—) and R³ being hydrogen, methyl or ethyl. Insome aspects, the heterologous moiety is a hyperbranched polyglyceroldescribed by the formula (R³—O—(CH₂—CHOR⁵—CH₂—O)_(n)—) with R₅ beinghydrogen or a glycerol chain described by the formula(R³—O—(CH₂—CHOR⁶—CH₂—O)_(n)—), with R⁶ being hydrogen or a glycerolchain described by the formula (R³—O—(CH₂—CHOR⁷—CH₂—O)_(n)—), with R⁷being hydrogen or a linear glycerol chain described by the formula(R³—O—(CH₂—CHOH—CH₂—O)_(n)—) and R³ being hydrogen, methyl or ethyl.Hyperbranched glycerol and methods for its synthesis are known in theart. See Oudshorn M, et al., Biomaterials 2006; 27:5471-5479, Wilms D,et al., Acc Chem Res 2010; 43:129-141 and references cited therein.

In certain aspects, the PG has a molar mass between 100 g/mol and 3000g/mol, particularly between 100 g/mol and 2500 g/mol, more particularlyof approx. 100 g/mol to 2000 g/mol. In certain aspects, the PG has amolar mass between 200 g/mol and 3000 g/mol, particularly between 300g/mol and 2500 g/mol, more particularly of approx. 400 g/mol to 2000g/mol.

In some aspects, the PG is PG₁₀₀, PG₂₀₀, PG₃₀₀, PG₄₀₀, PG₅₀₀, PG₆₀₀,PG₇₀₀, PG₅₀₀, PG₉₀₀, PG₁₀₀₀, PG₁₁₀₀, PG₁₂₀₀, PG₁₃₀₀, PG₁₄₀₀, PG₁₅₀₀,PG₁₆₀₀, PG₁₇₀₀, PG₁₈₀₀, PG₁₉₀₀, PG₂₀₀₀, PG₂₁₀₀, PG₂₂₀₀, PG₂₃₀₀, PG₂₄₀₀,PG₂₅₀₀, PG₁₆₀₀, PG₁₇₀₀, PG₁₈₀₀, PG₁₉₀₀, PG₂₀₀₀, PG₂₁₀₀, PG₂₂₀₀, PG₂₃₀₀,PG₂₄₀₀, PG₂₅₀₀, PG₂₆₀₀, PG₂₇₀₀, PG₂₅₀₀, PG₂₉₀₀, or PG₃₀₀₀. In oneparticular aspect, the PG is PG₄₀₀. In another particular aspect, the PGis PG₂₀₀₀.

In other particular aspects R₃ and/or R₇ of the compound of formula I isa radical of formula VI where R₁₃ is H and n is an integer from 9 to 45.In other particular aspects R₃ and R₇ of the compound of formula I is aradical of formula VI where R₁₃ is H and n is an integer from 9 to 45and R₁, R₅, R₉ and R₁₁ are all a radical of formula II. In otherparticular aspects the compound of formula I is a sodium salt with R₃and R₇=radical of formula VI where R₁₃ is H and n is an integer from 9to 45, and R₁, R₅, R₉ and R₁ are all a radical of formula II.

3. Pharmaceutical Compositions

In another aspect, the present invention also refers to pharmaceuticalcompositions comprising a compound, as defined in any of the aspectsdisclosed above. In some aspects, the pharmaceutical compositioncomprises a compound, as defined in any of the aspects disclosed above,together with one or more pharmaceutically acceptable excipients orcarriers. These pharmaceutical compositions are for use in increasingtissular perfusion and/or oxygenation in a subject in need thereof. Insome aspects, these pharmaceutical compositions are for use in thetreatment or prevention of ischemia and/or an ischemia-related diseaseor condition. In some aspects, the pharmaceutical compositions of thepresent invention are used for the treatment or prevention of PAD orCLI.

The term “excipient” as used herein refers to a substance which helpsabsorption of the elements of the pharmaceutical composition, stabilizessaid elements, activates or helps preparation of the composition. Thus,examples of excipients used in parenteral formulations include, but arenot limited to, antimicrobial agents (e.g., benzalkonium chloride,metacresol, thimerosal), co-solvents (e.g., ethanol), buffers and pHadjusting factors (e.g., carbonate, citrate, phosphate solutions).

As is the case for the excipient, the “pharmaceutically acceptablevehicle” is a substance used in the composition to dilute any of thecomponents contained therein to a determined volume or weight. Thepharmaceutically acceptable vehicle is an inert substance or a substancewith an analogous action to any of the elements comprising thepharmaceutical composition of the present invention. The role of saidvehicle is to allow the incorporation of other elements, allow betterdosing and administration or to provide consistency and shape to thecomposition.

Pharmaceutical compositions can comprise from approximately 1% toapproximately 95% of the compound as defined in any of the aspectsdisclosed above. In some aspects, the pharmaceutical compositions of thepresent invention can comprise, for instance, from approximately 20% toapproximately 90%, or from 20% to 80%, or from 20% to 70%, or from 20%to 60%, or from 20% to 50%, or from 30% to 90%, or from 40% to 90%, orfrom 50% to 90%, or from 60% to 90%, or from 30% to 70% of the compoundas defined in any of the aspects disclosed above.

In some aspects, the concentration of inositol phosphate of the presentinvention (e.g., myo-inositol hexaphosphate or an analog or derivativethereof, or a combination thereof) in each dose of the pharmaceuticalcomposition is between about 12.5 mM and about 135 mM. In some versionsof this aspect, the concentration of inositol phosphate of the presentinvention (e.g., myo-inositol hexaphosphate or an analog or derivativethereof, or a combination thereof) in each dose of the pharmaceuticalcomposition is about 25 mM, about 39 mM or about 114 mM.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise a compound as defined in any of the aspectsdisclosed above mixed with a pharmaceutically acceptable carrier (e.g.,as sterile water or sterile isotonic saline solution). Such formulationscan be prepared, packaged, or sold in a form suitable for bolusadministration or for continuous administration. Injectable formulationscan be prepared, packaged, or sold in unit dosage form, such as inampules or in multi-dose containers containing a preservative.Formulations for parenteral administration include, but are not limitedto, suspensions, solutions, emulsions in oily or aqueous vehicles,pastes, and implantable sustained-release or biodegradable formulations.Such formulations can further comprise one or more additionalingredients including, but not limited to, suspending, stabilizing, ordispersing agents.

In some aspects, in a formulation for parenteral administration, theactive agent (e.g., a compound as defined in any of the aspectsdisclosed above) is provided in dry (i.e., powder or granular) form forreconstitution with a suitable vehicle (e.g., sterile pyrogen-freewater) prior to parenteral administration of the reconstitutedcomposition.

The pharmaceutical compositions can be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution can be formulated according to the knownart, and may comprise, in addition to the active agent (e.g., compoundas defined in any of the aspects disclosed above), additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations can beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butanediol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides.

Other parentally-administrable formulations which are useful includethose which comprise the active agent (e.g., compound as defined in anyof the aspects disclosed above) in microcrystalline form, in a liposomalpreparation, or as a component of a biodegradable polymer system.

Compositions for sustained release or implantation can comprisepharmaceutically acceptable polymeric or hydrophobic materials such asan emulsion, an ion exchange resin, a sparingly soluble polymer, or asparingly soluble salt.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the present invention can be made using conventionaltechnology. In some cases, the dosage forms to be used can be providedas slow or controlled-release of one or more active agents thereinusing, for example, hydropropylmethyl cellulose, other polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, or microspheres or a combination thereof toprovide the desired release profile in varying proportions. Suitablecontrolled-release formulations known in the art, including thosedescribed herein, can be readily selected for use with thepharmaceutical compositions of the invention. Thus, single unit dosageforms suitable for parenteral or topical administration, such asinjectable solutions, gels, creams, and ointments, which are adapted forcontrolled-release are encompassed by the present invention.

Most controlled-release pharmaceutical products have a common goal ofimproving therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of therapeutic agent being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the therapeutic agent, reduceddosage frequency, and increased patient compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood level of thetherapeutic agent, and thus can affect the occurrence of side effects.

Most controlled-release formulations are designed to initially releasean amount of therapeutic agent that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of therapeutic agent to maintain this level of therapeuticeffect over an extended period of time. In order to maintain thisconstant level of therapeutic agent in the body, the therapeutic agentmust be released from the dosage form at a rate that will replace theamount of therapeutic agent being metabolized and excreted from thebody.

Controlled-release of an active agent can be stimulated by variousinducers, for example pH, temperature, enzymes, water, or otherphysiological conditions or compounds. The term “controlled-releasecomponent” in the context of the present invention is defined herein asa compound or compounds, including, but not limited to, polymers,polymer matrices, gels, permeable membranes, liposomes, or microspheresor a combination thereof that facilitates the controlled-release of theactive agent.

In certain aspects, the formulations of the present invention can be,but are not limited to, short-term, rapid-offset, as well as controlled,for example, sustained release, delayed release and pulsatile releaseformulations.

The term sustained release is used in its conventional sense to refer totherapeutic agent formulation (e.g., compound as defined in any of theaspects disclosed above)) that provides for gradual release of atherapeutic active agent over an extended period of time, and that can,although not necessarily, result in substantially constant blood levelsof a therapeutic agent over an extended time period. The period of timecan be as long as a month or more and should be a release which islonger that the same amount of agent administered in bolus form.

For sustained release, the compounds may be formulated with a suitablepolymer or hydrophobic material which provides sustained releaseproperties to the compounds. As such, the compounds for use the methodof the present invention can be administered in the form ofmicroparticles, for example, by injection or in the form of wafers ordiscs by implantation. In certain aspects, the compounds of theinvention are administered to a patient, alone or in combination withanother pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense torefer to a therapeutic agent formulation that provides for an initialrelease of the therapeutic agent after some delay following therapeuticagent administration. The delay may be from about 10 minutes up to about12 hours. The term pulsatile release is used herein in its conventionalsense to refer to a therapeutic agent formulation that provides releaseof the therapeutic agent in such a way as to produce pulsed plasmaprofiles of the therapeutic agent after administration. The termimmediate release is used in its conventional sense to refer to atherapeutic agent formulation that provides for release of thetherapeutic agent immediately after administration.

Additional formulations and dosage forms of the compositions of thepresent invention include dosage forms as described in U.S. Pat. Nos.6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790;US20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and20020051820; WO 2003035041, WO2003035040, WO2003035029, WO200335177,WO2003035039, WO2002096404, WO2002032416, WO2001097783, WO2001056544,WO2001032217, WO1998055107, WO1998011879, WO1997047285, WO1993018755,and WO1990011757.

Medicaments according to the invention are manufactured by methods knownin the art, especially by conventional mixing, coating, granulating,dissolving or lyophilizing.

The present invention also provides a compound, a combination ofcompounds, or pharmaceutical formulation as defined in any of the aboveaspects of the invention, in the broadest definition given, or asspecified in any of the aspects presented above, for use as amedicament.

4. Methods and Routes of Administration

In some aspects, the compound, pharmaceutical composition or combinedpreparation as defined in any of the aspects disclosed above isadministered jointly, concurrently or sequentially with anothertherapeutic agent. In some versions of this aspect, the additionaltherapeutic agent comprises cilostazol, pentoxifylline or combinationthereof.

In some aspects, the administration of an effective amount of compound,pharmaceutical composition or combined preparation as defined in any ofthe aspects above is provided. Said compound, pharmaceutical compositionor combined preparation can be administered parenterally such as, forexample, intravenously, intraperitoneally, intramuscularly,intra-arterially, intradermal, intrathecal, epidural or spinal orsubcutaneously. The parenteral administration may be by bolus injectionor by intravenous infusion.

In a particular aspect of the present invention, myo-inositolhexaphosphate (or a formulation comprising myo-inositol hexaphosphatesuch as SNF472) is administered via intravenous infusion. In anotherparticular aspect of the present invention, myo-inositol hexaphosphateis administered subcutaneously. In another aspect a derivative ofinositol or myo-inositol hexaphosphate derivative, for example, acompound of formula I with R₃ and R₇=radical of formula V where R₁₃ is Hand n is an integer from 2 to 200, and R₁, R₅, R₉ and R₁₁ are all aradical of formula II (or a sodium salt thereof) is administered viaintravenous infusion. In another aspect, a derivative of inositol ormyo-inositol hexaphosphate derivative, for example, a compound offormula I with R₃ and R₇=radical of formula V where R₁₃ is H and n is aninteger from 2 to 200, and R₁, R₅, R₉ and R₁₁ are all a radical offormula II (or a sodium salt thereof) is administered subcutaneously.

Alternatively, the compound, pharmaceutical composition or combinedpreparation can be administered as a component of a hemodialysis,hemofiltration, or peritoneal dialysis solution or system.

In the particular case of patients treated with dialysis, a veryappropriate method of administration consists of an administration(e.g., a non-bolus type administration) of an inositol phosphate of thepresent invention via the dialysis apparatus (before or after thefilter) instead of directly injecting the inositol phosphate of thepresent invention into the patient intravenously. Thus, blood can betreated with the inositol phosphate of the present invention (e.g.,myo-inositol hexaphosphate) as it leaves the patient and circulatesthrough the dialysis circuit and, when the blood containing the inositolphosphate of the present invention returns to the body.

Thus, in some aspects, the compound, pharmaceutical composition orcombined preparation, as defined in any of the aspects disclosed above,is administered to a patient during hemodialysis. In some aspects, thecompound, pharmaceutical composition or combined preparation, as definedin any of the aspects disclosed above, is administered to the bloodextracted from the patient during hemodialysis, preferably before it isfiltered (i.e., the therapeutic agent is administered to the patient'sunfiltered blood in the dialysis circuit). In some aspects, the compoundis inositol hexaphosphate, in particular myo-inositol hexaphosphatesodium salt or a derivative thereof, namely, a compound of formula Iwith R₃ and R₇=radical of formula V where R₁₁ is H and n is an integerfrom 2 to 200, and R₁, R₅, R₉ and R₁₁ are all a radical of formula II(or a sodium salt thereof).

In the case of dialysis patients, administration of an inositolphosphate of the present invention (e.g., myo-inositol hexaphosphate)via the dialysis apparatus allows the blood to equilibrate with thedialysis fluid prior to returning to the body; thus, although inositolphosphate of the present invention (e.g., myo-inositol hexaphosphate)can sequester ionic calcium, this fact is compensated when the bloodpasses through the dialysis filter thereby eliminating said side effectand significantly improving the safety profile. Additionally,administering the inositol phosphate of the present invention (e.g.,myo-inositol hexaphosphate) concomitant to hemodialysis, in particularwhen administered to the unfiltered blood extracted from the patientduring hemodialysis, allows for reducing the dose of the compound withconsequent advantages in terms of reduced toxicity and minimizingadverse side effects.

In some aspects, the compound, pharmaceutical composition or combinedpreparation as defined in any of the aspects disclosed above isadministered to a patient that is being treated with hemodialysis beforethe dialysis treatment or after a dialysis treatment.

In general, an effective dose of an inositol phosphate of the presentinvention (e.g., myo-inositol hexaphosphate) administered according tothe methods disclosed herein will depend, for example, on the relativeefficacy of the compound concerned, the severity of the disordertreated, and the species and weight of the subject. In some aspects, theeffective dose of an inositol phosphate of the present invention for asubject of a certain species (e.g., human) can be calculated based onthe experimental data available for a different or reference species(e.g., rat).

Thus, for example, a dose of inositol myo-hexaphosphate administered aspart of a regimen comprising the administration of a dosage of 20 mg/kgto a rat subject would be equivalent to administering the same activeagent at a dosage of 4.2 mg/kg to a human subject (i.e., a total dose of300 mg of inositol myo-hexaphosphate to a human subject weighingapproximately 70 kg). Likewise, a dosage of 40 mg/kg to a rat subjectwould be equivalent to administering inositol myo-hexaphosphate at adosage of 8.4 mg/kg to a human subject as defined before. The dosagescan be adjusted based on the subjects age, species, weight, bodysurface, renal clearance, sex, pathological state, route ofadministration, concurrent administration of one or more other drugs,and a wide variety of physiologic and psychological factors usingmethods known in the art (Pan S., et al., Patient Prefer Adherence 2016;10:549-560; Pai M, Pharmacotherapy 2012; 32:856-868; Hacker M., et al.,Eds, “Pharmacology: Principles and Practice” (Academic Press;Burlington, Mass., USA, 2009). The term “mg/kg” as used herein refers tomg of an inositol phosphate of the present invention per kilogram of thebody mass (body weight) of the subject.

In some aspects, the dose of inositol phosphate of the present invention(e.g., myo-inositol hexaphosphate) comprises from about 0.001 mg/kg toabout 60 mg/kg of an inositol phosphate, an inositol phosphate analog,an inositol phosphate derivative, or combination thereof according thepresent invention. In some further aspects, the dose of inositolphosphate of the present invention (e.g., myo-inositol hexaphosphate) isbetween about 0.001 mg/kg and about 20.0 mg/kg, between about 20.0 mg/kgand about 40.0 mg/kg, or between about 40.0 mg/kg and about 60.0 mg/kg.

In some aspects, the dose of inositol phosphate of the present invention(e.g., myo-inositol hexaphosphate) is between about 0.001 mg/kg andabout 1.0 mg/kg, between about 1.0 mg/kg and about 10.0 mg/kg, betweenabout 10.0 mg/kg and about 20.0 mg/kg, between about 20.0 mg/kg andabout 30.0 mg/kg, between about 30.0 mg/kg and about 40.0 mg/kg, betweenabout 40.0 mg/kg and about 50.0 mg/kg, or between about 50.0 mg/kg andabout 60.0 mg/kg.

In some aspects, the dose of inositol phosphate of the present invention(e.g., myo-inositol hexaphosphate) is between about 0.001 mg/kg andabout 0.5 mg/kg, between about 0.5 mg/kg and about 1.0 mg/kg, betweenabout 1.0 mg/kg and about 5.0 mg/kg, between about 5.0 mg/kg and about10.0 mg/kg, between about 10.0 mg/kg and about 15.0 mg/kg, between about15.0 mg/kg and about 20.0 mg/kg, between about 20.0 mg/kg and about 25.0mg/kg, between about 25.0 mg/kg and about 30.0 mg/kg, between about 30.0mg/kg and about 35.0 mg/kg, between about 35.0 mg/kg and about 40.0mg/kg, between about 40.0 mg/kg and about 45.0 mg/kg, or between about45.0 mg/kg and about 50.0 mg/kg.

In some aspects, the dose of inositol phosphate of the present invention(e.g., myo-inositol hexaphosphate) is between about 0.001 mg/kg andabout 0.25 mg/kg, between about 0.25 mg/kg and about 0.5 mg/kg, betweenabout 0.5 mg/kg and about 0.75 mg/kg, between about 0.75 mg/kg and about1.0 mg/kg, between about 1.0 mg/kg and about 2.50 mg/kg, between about2.50 mg/kg and about 5.0 mg/kg, between about 5.0 mg/kg and about 7.5mg/kg, between about 7.5 mg/kg and about 10.0 mg/kg, between about 10.0mg/kg and about 12.5 mg/kg, between about 12.5 mg/kg and about 15.0mg/kg, between about 15.0 mg/kg and about 17.5 mg/kg, between about 17.5mg/kg and about 20.0 mg/kg, between about 20.0 mg/kg and about 22.5mg/kg, between about 22.5 mg/kg and about 25.0 mg/kg, between about 25.0mg/kg and about 27.5 mg/kg, between about 27.5 mg/kg and about 30.0mg/kg, between about 30.0 mg/kg and about 32.5 mg/kg, between about 32.5mg/kg and about 35.0 mg/kg, between about 35.0 mg/kg and about 37.5mg/kg, between about 37.5 mg/kg and about 40.0 mg/kg, between about 40.0mg/kg and about 42.5 mg/kg, between about 42.5 mg/kg and about 45.0mg/kg, between about 45.0 mg/kg and about 47.5 mg/kg, between about 47.5mg/kg and about 50.0 mg/kg, between about 50.0 mg/kg and about 52.5mg/kg, between about 52.5 mg/kg and about 55.0 mg/kg, between about 55.0mg/kg and about 57.5 mg/kg, or between about 57.5 mg/kg and about 60.0mg/kg.

In some aspects, the dose of inositol phosphate of the present invention(e.g., myo-inositol hexaphosphate) is between about 0.25 mg/kg and about60.0 mg/kg, between about 0.5 mg/kg and about 60.0 mg/kg, between about0.75 mg/kg and about 60.0 mg/kg, between about 1.0 mg/kg and about 60.0mg/kg, between about 2.50 mg/kg and about 60.0 mg/kg, between about 5.0mg/kg and about 60.0 mg/kg, between about 7.5 mg/kg and about 60.0mg/kg, between about 10.0 mg/kg and about 60.0 mg/kg, between about 12.5mg/kg and about 60.0 mg/kg, between about 15.0 mg/kg and about 60.0mg/kg, between about 17.5 mg/kg and about 60.0 mg/kg, between about 20.0mg/kg and about 60.0 mg/kg, between about 22.5 mg/kg and about 60.0mg/kg, between about 25.0 mg/kg and about 60.0 mg/kg, between about 27.5mg/kg and about 60.0 mg/kg, between about 30.0 mg/kg and about 60.0mg/kg, between about 32.5 mg/kg and about 60.0 mg/kg, between about 35.0mg/kg and about 60.0 mg/kg, between about 37.5 mg/kg and about 60.0mg/kg, between about 40.0 mg/kg and about 60.0 mg/kg, between about 42.5mg/kg and about 60.0 mg/kg, between about 45.0 mg/kg and about 60.0mg/kg, between about 47.5 mg/kg and about 60.0 mg/kg, between about 50.0mg/kg and about 60.0 mg/kg, between about 52.5 mg/kg and about 60.0mg/kg, between about 55.0 mg/kg and about 60.0 mg/kg, or between about57.5 mg/kg and about 60.0 mg/kg.

In some aspects, the dose of inositol phosphate of the present invention(e.g., myo-inositol hexaphosphate) is between about 0.001 mg/kg andabout 57.5 mg/kg, between about 0.001 mg/kg and about 55.0 mg/kg,between about 0.001 mg/kg and about 52.5 mg/kg, between about 0.001mg/kg and about 50.0 mg/kg, between about 0.001 mg/kg and about 47.5mg/kg, between about 0.001 mg/kg and about 45.0 mg/kg, between about0.001 mg/kg and about 42.5 mg/kg, between about 0.001 mg/kg and about40.0 mg/kg, between about 0.001 mg/kg and about 37.5 mg/kg, betweenabout 0.001 mg/kg and about 35.0 mg/kg, between about 0.001 mg/kg andabout 32.5 mg/kg, between about 0.001 mg/kg and about 30.0 mg/kg,between about 0.001 mg/kg and about 27.5 mg/kg, between about 0.001mg/kg and about 25.0 mg/kg, between about 0.001 mg/kg and about 22.5mg/kg, between about 0.001 mg/kg and about 20.0 mg/kg, between about0.001 mg/kg and about 27.5 mg/kg, between about 0.001 mg/kg and about25.0 mg/kg, between about 0.001 mg/kg and about 22.5 mg/kg, betweenabout 0.001 mg/kg and about 20.0 mg/kg, between about 0.001 mg/kg andabout 17.5 mg/kg, between about 0.001 mg/kg and about 15.0 mg/kg,between about 0.001 mg/kg and about 12.5 mg/kg, between about 0.001mg/kg and about 10.0 mg/kg, between about 0.001 mg/kg and about 7.5mg/kg, between about 0.001 mg/kg and about 5.0 mg/kg, or between about0.001 mg/kg and about 2.5 mg/kg.

5. Indications

The compounds, pharmaceutical compositions, combined preparations,methods and routes of administration, as defined in any of the aspectsdisclosed above, can be used for increasing tissular perfusion and/oroxygenation in a subject in need thereof.

The terms “ischemia-related disease or condition” as used herein referto any diseases or condition related to or arising from an ischemicevent or injury. Examples of ischemia-related diseases or conditionsinclude, but are not limited, to cerebrovascular (e.g., stroke,transient ischemic attack (TIA), subarachnoid hemorrhage, vasculardementia), cardiovascular (e.g., myocardial infarction, anginapectoris), gastrointestinal (e.g., colitis), peripheral (e.g., acutelimb ischemia) and cutaneous (e.g., cyanosis, gangrene) diseases orconditions.

In some aspects, the compounds, pharmaceutical compositions, combinedpreparations, methods and routes of administration of the presentinvention can be used for the treatment or prevention of ischemia and/oran ischemia-related disease or condition in a subject in need thereof.

The term “kidney failure” as used herein refers to is a disease thatcauses a progressive loss of kidney function, with a concomitantdecrease in the glomerular filtration rate (GFR) or index. Kidneyfailure is also known as renal impairment or kidney disease. Kidneydisease can be classified as (i) acute kidney injury (AKI), aprogressive loss of kidney function, which generally causes oliguria anda fluid and electrolyte imbalance and (ii) chronic kidney disease (CKD),a much slower loss of kidney function over a period of months or years.Depending on the degree of kidney function, five stages of CKD aredefined on the basis of the GFR: (a) stage 1, normal or high GFR (>90ml/min), (b) stage 2: Mild CKD, GFR=60-89 mi/min, (c) stage 3, moderateCKD, GFR=30-59 m/min, (d) stage 4, severe CKD, GFR=15-29 ml/min and (e)stage 5, terminal CKD, GFR<15 ml/min. In stage 5, dialysis or a kidneytransplant are required to maintain the state of health. AKI and CKD mayoccur concomitantly, which is known as acute-on-chronic renal failure.

In some aspects, the compounds, pharmaceutical compositions, combinedpreparations, methods and routes of administration of the presentinvention can be used to increase tissular perfusion in a subject withkidney disease. The kidney disease in the subject can be acute, chronicor both. In some aspects, the subject is undergoing dialysis (e.g.,peritoneal, hemodialysis). In a further form of this aspect, the subjectis undergoing hemodialysis. In some other aspects, the subject is notdialyzed (e.g., a subject with CKD in stages 1 to 4). In one version ofthis aspect, the subject is administered an inositol phosphate of thepresent invention (e.g., myo-inositol hexaphosphate) in an effectivedosage of about 0.001 mg/kg to about 60 mg/kg.

In some aspects, the compounds, pharmaceutical compositions, combinedpreparations, methods and routes of administration of the presentinvention can be used for treating or preventing ischemia and/or anischemia-related disease or condition in a subject with kidney disease.The kidney disease in the subject can be acute, chronic or both. In oneversion of this aspect, the subject is undergoing dialysis (e.g.,peritoneal, hemodialysis). In a further form of this version, thesubject is undergoing hemodialysis. In another version of this aspect,the subject is not dialyzed (e.g., a subject with CKD in stages 1 to 4).In one version of this aspect, the subject is administered an inositolphosphate of the present invention (e.g., myo-inositol hexaphosphate) inan effective dosage of about 0.001 mg/kg to about 60 mg/kg.

In some aspects, the compounds, pharmaceutical compositions, combinedpreparations, methods and routes of administration of the presentinvention can be used for improving the walking ability of a subject inneed thereof. In some aspects, the compounds, pharmaceuticalcompositions, methods and routes of administration of the presentinvention can be used for increasing the Maximal Walking Distance (MWD),Maximal Walking Time (MWT) or both in a subject in need thereof. In someaspects, the subject is affected with kidney disease. The kidney diseasein the subject can be acute, chronic or both. In one version of thisaspect, the subject is undergoing dialysis (e.g., peritoneal,hemodialysis). In a further form of this version, the subject isundergoing hemodialysis. In another version of this aspect, the subjectis not dialyzed (e.g., a subject with CKD in stages 1 to 4). In oneversion of this aspect, the subject is administered an inositolphosphate of the present invention (e.g., myo-inositol hexaphosphate) inan effective dosage of about 0.001 mg/kg to about 60 mg/kg.

The compounds, pharmaceutical compositions, combined preparations,methods and routes of administration of the present invention areparticularly useful for increasing tissular perfusion and/or oxygenationin the lower limbs and, especially, for the treatment and prevention ofperipheral artery disease. A further condition that can benefit from theuse of the inositol phosphates of the present invention is critical limbischemia. In particular aspects the compounds, pharmaceuticalcompositions, combined preparations, methods and routes ofadministration as defined in any of the aspects disclosed above are foruse in increasing tissular perfusion and/or oxygenation, especially, inthe lower limbs and, especially, for the treatment and prevention of PADand/or CLI. In some aspects, the subject is undergoing dialysis (e.g.,peritoneal, hemodialysis). In some further aspects, the subject isundergoing hemodialysis. In some other aspects, the subject is notdialyzed (e.g., a subject with CKD in stages 1 to 4). In one version ofthis aspect, the subject is administered an inositol phosphate of thepresent invention (e.g., myo-inositol hexaphosphate) in an effectivedosage of about 0.001 mg/kg to about 60 mg/kg.

The following embodiments further illustrate the scope of the invention:

Embodiment 1. A compound of general formula I, or a pharmaceuticallyacceptable salt thereof, for use in increasing tissular perfusion and/oroxygenation in a subject in need thereof

where R₁, R₃, R₅, R₇, R₉ and R₁₁ are independently selected from OH, aradical of formula II, III, IV, V, VI and VII:

wherein: n is an integer in the range from 2 to 200, and R₁₃ is selectedfrom H, methyl, ethyl and C₃-C₁₀ alkyl;with the condition that:at least one of R₁, R₃, R₅, R₇, R₉ and R₁₁ is selected from a radical offormula II, III and IV, andzero, one, two or three of R₁, R₃, R₅, R₇, R₉ and R₁₁ is selected from aradical of formula V, VI and VII.

Embodiment 2. The compound for use according to embodiment 1, that isfor the treatment or prevention of peripheral arterial disease.

Embodiment 3. The compound for use according to any of the precedingembodiments, that is for the treatment or prevention critical limbischemia.

Embodiment 4. The compound for use according to any of the precedingembodiments, that is for treating a subject that is being subjected todialysis, preferably hemodialysis.

Embodiment 5. The compound for use according to any of the precedingembodiments, that is a sodium salt.

Embodiment 6: The compound for use according to any of the precedingembodiments, wherein at least two, at least three, at least four, atleast five or at least 6 of R₁, R₃, R₅, R₇, R₉ and R₁₁ are a selectedfrom the radicals of formulas V, VI and VII.

Embodiment 7. The compound for use according to the precedingembodiment, wherein at least two, at least three, at least four, atleast five or at least six of R₁, R₃, R₅, R₇, R₉ and R₁₁ are a radicalof formula V.

Embodiment 8. The compound for use according to the precedingembodiment, wherein the compound of formula I is inositol hexaphosphate.

Embodiment 9. The compound for use according to the precedingembodiment, that is a hexasodium salt.

Embodiment 10. The compound for use according to embodiment 7, wherein:

R₇ is OSO₃ ⁻, and R₁, R₃, R₅, R₉ and R₁₁ are independently selected fromOPO₃ ²⁻, OPSO₂ ²⁻ or OSO₃ ⁻.R₉, R₅ and R₁ are OPO₃ ²⁻ and R₇, R₃ and R₁ are OSO₃ ⁻;R₉, R₅ and R₁ are OSO₃ ⁻ and R₇, R₃ and R₁ are OPO₃ ²⁻;R₃, R₁ and R₁ are OSO₃ ⁻ and R₉, R₇ and R₅ are OPO₃ ²⁻;R₃, R₁ and R₁ are OPO₃ ²⁻ and R₉, R₇ and R₅ are OSO₃ ⁻;R₇ and R₁ are OPO₃ ²⁻ and R₉, R₅, R₃, and R₁ are OPO₃ ⁻;R₇ and R₁ are OSO₃ ⁻ and R₉, R₅, R₃, and R₁ are OPO₃ ²⁻.R₇ and R₅ are OPO₃ ²⁻ and R₉, R₃, R₁, and R₁ are OSO₃ ⁻; or,R₇ and R₅ are OSO₃ ⁻ and R₉, R₃, R₁, and R₁ are OPO₃ ²⁻

Embodiment 11. The compound for use according to any of the precedingembodiments, wherein the compound of formula I has myo-inositolconformation.

Embodiment 12. The compound for use according to any one of embodiments1-6, wherein one, two or three of R₁, R₃, R₅, R₇, R₉ and R₁₁ is selectedfrom a radical of formula V, VI and VII.

Embodiment 13. The compound for use according to the precedingembodiment, wherein four of R₁, R₃, R₅, R₇, R₉ and R₁₁ are a radical offormula II and two of R₁, R₃, R₅, R₇, R₉ and R₁₁ are selected from aradical of formulas V, VI and VII.

Embodiment 14. The compound for use according to the precedingembodiment, wherein four of R₁, R₃, R₅, R₇, R₉ and R₁₁ are a radical offormula II and two of R₁, R₃, R₅, R₇, R₉ and R₁₁ are a radical offormula V.

Embodiment 15. The compound for use according to any one of embodiments12-14, wherein:

(i) R₁, R₅, R₉ and R₁₁ are a radical of formula II and R₃ and R₇ areselected from a radical of formulas V, VI and VII,(ii) R₁, R₃, R₉ and R₁₁ are a radical of formula II and R₅ and R₇ areselected from a radical of formulas V, VI and VII.

Embodiment 16. The compound for use according to the precedingembodiment, wherein the radical selected from V, VI and VII is theradical of formula V.

Embodiment 17. The compound for use according to any of the embodiments12-16, wherein the radical of formula V, VI or VI has n in the rangefrom 2 to 200.

Embodiment 18. The compound for use according to the precedingembodiment, wherein n is the range from 9 to 30.

Embodiment 19. The compound for use according to the precedingembodiment, wherein n is the range from 15 to 30.

Embodiment 20. The compound for use according to embodiment 17, whereinn is the range from 3 to 9.

Embodiment 21. The compound for use according to any of the embodiments12-20, wherein R₁₃ is H.

Embodiment 22. The compound for use according to embodiment 21, whereinR₁, R₅, R₉ and R₁₁ are a radical of formula II and R₃ and R₇ are aradical of formula V.

Embodiment 23. A pharmaceutical composition for the use as defined inany of the embodiments 1-4 comprising the compound as defined in any ofthe preceding embodiments together with pharmaceutically acceptableexcipients and carriers.

Embodiment 24. The pharmaceutical composition according to the previousembodiment, wherein the compound is present at 20 to 90% (w/w) of thetotal composition.

Embodiment 25. The pharmaceutical composition according to the previousembodiment, wherein the compound is present at 30 to 80% (w/w) of thetotal composition.

Embodiment 26. The pharmaceutical composition according to the previousembodiment, wherein the compound is present at 40 to 70% (w/w) of thetotal composition.

Embodiment 27. The pharmaceutical composition according to any of theembodiments 23-26, wherein the composition is in dry form forreconstitution with a suitable vehicle.

Embodiment 28. The pharmaceutical composition according to any of theembodiments 23-26, wherein the composition is in solution, preferablyisotonic saline solution.

Embodiment 29. The pharmaceutical composition according to any of theembodiments 23-27, that forms part of a hemodialysis, hemofiltration, orperitoneal dialysis solution.

Embodiment 30. The pharmaceutical composition according to any of theembodiments 23-29, wherein the composition is for controlled release.

Embodiment 31. The compound for use according to any one of theembodiments 1-22 or the pharmaceutical composition for use according toany of the embodiments 23-30, that is administered to a patient that isbeing subjected to dialysis.

Embodiment 32. The compound for use according to the previousembodiment, wherein the dialysis is hemodialysis.

Embodiment 33. The compound or pharmaceutical composition for useaccording to any of the embodiments 31-32, that is administered beforethe dialysis.

Embodiment 34. The compound or pharmaceutical composition for useaccording to any of the embodiments 31-32, that is administered duringthe dialysis.

Embodiment 35. The compound or pharmaceutical composition for useaccording to any of the embodiments 31-32, that is administered afterthe dialysis.

Embodiment 36. The compound or pharmaceutical composition for useaccording to any of the embodiments 31-35, that is administered byparenteral route.

Embodiment 37. The compound or pharmaceutical composition for useaccording to the preceding embodiment wherein the parenteraladministration is intravenous, subcutaneous or intramuscular.

Embodiment 38. The compound or pharmaceutical composition for useaccording to the preceding embodiment, wherein the intravenousadministration is by bolus injection or by intravenous infusion.

Embodiment 39. The compound or pharmaceutical composition for useaccording to embodiment 34, that is administered to the unfiltered bloodextracted from the patient.

Embodiment 40. The compound or pharmaceutical composition for useaccording to any of the preceding embodiments, wherein the compound isadministered to the subject in a therapeutically effective dosage ofabout 0.001 mg/kg to about 60 mg/kg.

Embodiment 41. The compound or pharmaceutical composition for useaccording to embodiment 40, wherein the compound is administered to thesubject in a therapeutically effective dosage of about 15 mg/kg to about45 mg/kg.

Embodiment 42. A combined preparation comprising: (i) (a) at least onecompound according to any one of the embodiments 1-22 or (b) at leastone pharmaceutical composition according to any of the embodiments23-30, and (ii) at least one additional therapeutic agent for use inhuman health.

Embodiment 43. The combined preparation according to the precedingembodiment wherein the additional therapeutic agent is cilostazol,pentoxifylline or a combination thereof.

Embodiment 44. A method for increasing tissular perfusion and/oroxygenation in a subject in need thereof which comprises administering atherapeutically effective amount of the compound or pharmaceuticalcomposition according to any one of embodiments 1-41 or the combinedpreparation according to any one of the embodiments 42-43 to thesubject.

Embodiment 45. A method for treating or preventing ischemia and/or anischemia-related disease or condition in a subject in need thereof whichcomprises administering a therapeutically effective amount of thecompound or pharmaceutical composition according to any one ofembodiments 1-41 or the combined preparation according to any one of theembodiments 42-43 to the subject.

Embodiment 46. A method for improving the walking ability in a subjectin need thereof which comprises administering a therapeuticallyeffective amount of the compound or pharmaceutical composition accordingto any one of embodiments 1-41 or the combined preparation according toany one of the embodiments 42-43 to the subject.

Embodiment 47. A method for increasing the Maximal Walking Distance(MWD), Maximal Walking Time (MWT) or both in a subject in need thereofwhich comprises administering a therapeutically effective amount of thecompound or pharmaceutical composition according to any one ofembodiments 1-41 or the combined preparation according to any one of theembodiments 42-43 to the subject.

Embodiment 48. A method for treating or preventing peripheral arterialdisease in a subject in need thereof which comprises administering atherapeutically effective amount of the compound or pharmaceuticalcomposition according to any one of embodiments 1-41 or the combinedpreparation according to any one of the embodiments 42-43 to thesubject.

Embodiment 49. The methods according to any one of embodiments 44-48wherein the combined preparation is administered jointly, concurrentlyor sequentially to the subject.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all documents citedthroughout this application are incorporated herein in their entirety byreference.

General Procedures

1. Limb Blood Perfusion

Position limb blood perfusion and ischemia status (i.e., blood perfusionincluding, perfusion unit, perfusion difference and perfusion ratio) isevaluated by laser doppler perfusion imaging using a PeriCam PSI NRimager (Perimed AB, Jarfalla, SE). Subjects are anesthetized using 3%isoflurane delivered in 100% oxygen at a flow rate at 1 L/min beforemeasuring. The perfusion difference and perfusion ratio are calculatedby comparing the baseline and any indicated interim or final readingsfor each group. Blood flux is assessed around the C_(max) of the testedactive agents (i.e., 15 min after treatment with SNF472, 20 min aftertreatment with IP4-BIS-PEG100, and 3 to 4 hours after treatment withcilostazol).

2. Walking Ability Tests

Quantification of walking ability (maximal waling time (MWT) and maximalwalking distance (MWD)) are measured by a forced incremental treadmillrunning test. A two-lane rodent treadmill (LE8709TS; PanLab/HarvardApparatus, Holliston, Mass., US) is employed. The treadmill ismaintained at a 15% inclination running at a speed of 15 m/min (25cm/sec) during the first 5 min, then at 33 cm/sec during the following 5min, and finally, at a speed of 40 cm/sec for a maximum of 30 additionalminutes.

Limb function is assessed around the C_(max) of the tested active agents(i.e., 15 min after treatment with SNF472, and 3 to 4 hours aftertreatment with cilostazol). Subjects are exercised in the treadmill foracclimatization according to a set protocol prior to testing. Subjectsthat do not comply with the protocol are excluded from the test.

Subjects are kept running for up to 40 min or until exhausted (i.e.,they remain on the shock grid for five continuous seconds) during thetest. MWD and MWT are then calculated for each animal.

3. Tissue and Blood Collection, Calcification Assays

Subjects are anesthetized by isoflurane inhalation. Blood is obtained byexsanguination through cardiac puncture. The subjects are thensacrificed and their tissues (e.g., right and left femoral arteries,aorta) are collected. The blood and tissues are processed and analyzedfor determining their calcium contents.

Calcium content in tissue sample is quantified via inductively coupledplasma optical emission spectrometry (ICP-OES) using an Optima 7300 DVICP-OES System spectrometer (PerkinElmer, Inc., Waltham, Mass., US)according to the manufacturer's instructions. Myo-inositol-hexaphosphatelevels in plasma are quantified by LC-MS/MS chromatographic methodsdescribed in the art. See WO2013050603.

Example 1 Prevention of Limb Ischemia Impact in Blood Perfusion

The preventive effects of SNF472, IP4-4,6-bisPEG100 sodium salt andcilostazol on blood perfusion were tested using a rat model for aduration of 12 days. Limb ischemia in the subjects was induced from D1excepting the sham group, to which no ischemia was induced. Subjectsinduced with ischemia were then treated with placebo and active agentformulations from D1 to D12 to assess their impact in preventing limbischemia. Observations were taken at several points during treatmentfrom D1 to D12. All subjects were weighed every day before treatment.

1. Induction of Limb Ischemia

Fifty-four male Sprague Dawley (SD) rats (Envigo Corp., Huntingdon, GB)weighing approximately 250-275 g were used. The subjects were fed withan A04 diet (Scientific Animal Food & Engineering; Carpe Bio,Amersfoort, NL). The subjects were divided in 5 groups, 8 to 10 rats pergroup, as follows:

Group 1—Control (sham)

Group 2a Placebo—Physiological saline solution

Group 2b Placebo—5% carboxymethyl cellulose (CMC) sodium salt solution

Group 3—SNF472 (Na₆IP₆)

Group 4—IP4-4,6-bisPEG100 sodium salt

Group 5—Cilostazol (C₂₀H₂₇N₅O₂)

Limb ischemia was induced in the subjects of groups 2a-5 by subcutaneousadministration of 120,000 IU/kg vitamin D3 (cholecalciferol, DuphafralD₃ 1000; Zoetis Inc., Parsippany, N.J., US) in physiological salinesolution (2 mL/kg) every day during D1 to D3. The sham group 1 subjectswere administered physiological saline solution (2 mL/kg) subcutaneouslyevery day during D1 to D3 with no vitamin D3.

The subjects in groups 1 and 2a were administered physiological salinesolution (2 mL/kg) subcutaneously every day during D1 to D12. Thesubjects in group 2b were administered a 5% CMC sodium salt in watersolution (5 mL/kg) orally every day during D1 to D12.

The administration of vitamin D3 induced ischemia in the posterior limbsof the subjects in groups 2a, 2b, and 3-5 was assayed by laser dopplerperfusion imaging.

2. Ischemia Rescue and Effects on Limb Blood Perfusion—Laser DopplerImaging Assay

Limb blood perfusion was induced in the subjects of groups 3 and 4 bysubcutaneous administration of 20 mg/kg of SNF472 (Na₆IP₆; free base:600 g/mol) and IP4-4,6-bisPEG100 sodium salt (free base 696.27 g/mol),respectively, in physiological saline solution (2 mL/kg) every dayduring D1 to D12. Limb blood perfusion was induced in the group 5subjects by oral administration of 20 mg/kg cilostazol (C₂₀H₂₇N₅O₂, freebase 369.46 g/mol; lot no. LRAB9590, Sigma-Aldrich Corp., St. Louis,Mo., US) in a 5% CMC sodium salt in water solution (5 mL/kg) every dayduring D1 to D12.

Limb ischemia status was evaluated at days D0 (baseline), D6 and D12 inall rats by laser doppler perfusion imaging. The perfusion differenceand perfusion ratio were calculated by comparing the D0 baseline andeither the D6 and D12 readings for each group. In particular, theperfusion difference and perfusion ratio were calculated by comparingthe group 1 (control) and (a) groups 2a and 2b placebo (i.e.,physiological saline solution, 5% CMC sodium salt in water solution),(b) group 3 (SNF472), (c) group 4 (IP4-4,6-bisPEG100 sodium salt), and(d) group 5 (cilostazol) readings.

On the days when blood perfusion tests involving the use of activeagents were conducted (i.e., D6, D12), the dosages were administered asfollows:

Group 3—SNF472 (Na₆IP₆): 15 minutes before reading

Group 4—IP4-4,6-bisPEG100 sodium salt: 20 minutes before reading

Group 5—Cilostazol (C₂₀H₂₇N₅O₂): 3 to 4 hours before reading

Under the scheme above, the active agents would be at their maximumserum concentration (C_(max)) at the time when the test readings weretaken.

The group 1 subjects did not show any significant changes in theirperfusion parameters.

The administration of SNF472 and the IP4-4,6-bisPEG100 sodium saltattenuated the drop in blood perfusion in the limbs of the subjects ofgroups 3-5 (i.e., treated with vitamin D3) compared to both placebo andcilostazol. These results suggest that SNF472 and IP4-4,6-bisPEG100 aremore effective than cilostazol for increasing blood perfusion in theposterior limbs of the treated subjects. See FIGS. 7, 8, and 9.

3. Calcium Content and Calcification—ICP-OES

SNF472 showed to be effective against vascular calcification, as SNF472inhibited aorta calcification (31±16%) after a daily subcutaneous dosingof 20 mg/kg compared to placebo. Cilostazol was not active againstcalcification at the same dose. See FIG. 10.

4. Pharmacokinetics

The subjects from groups 1, 2, 3 and 5 were sacrificed afterexsanguination. Then, their necropsies were performed, and their aortaswere collected. The tissues were lyophilized for 24 h and weighed. Thelyophilized tissues were then digested using a 1:1 HNO₃:HClO₄ mixture ina dry bath incubator for 2-4 h at 180° C. The digested tissues weresubsequently diluted using ultra-pure Milli-Q water (MilliporeSigma(Merck KGaA), Burlington, Mass., US) to a final volume of 10 mL. Thecalcium content in the aorta sample was quantified via ICP-OES.

Subjects from groups 1, 2, 3, 4 and 5 were anesthetized and their bloodwas obtained in D12. Around 8-10 mL of total blood per subject weretaken and divided in collection tubes for plasma (K3EDTA, approximately6 mL blood) and serum (approximately 2-3 mL blood) testing. Plasma wasstored in one aliquot of 600 μL and several other aliquots of 500 μL.Serum was divided in two aliquots.

Myo-inositol-hexaphosphate levels in the plasma of the subjects of group3 (i.e., taken at around C_(max), 15 min after the last SNF472 dosing)were quantified by LC-MS/MS chromatographic methods described in theart. See WO2013050603.

All subjects in group 3 were well exposed to the SNF472 product. Theplasma level 15 min after the last subcutaneous dosing in D12 was15587±ng/mL (24±12 μM). The plasma levels in rats after a daily dosingat 20 mg/kg were comparable to the levels found in hemodialysis patientstreated with SNF472 at a dose of 4.2 mg/kg via the intravenous route.

Example 2 Prevention of Limb Ischemia Impact on Maximal Walking (MWT)and Distance (MD T) Times

The preventive effects of SNF472 and cilostazol on blood perfusion,walking ability and tissue calcification were tested using a rat modelfor a duration of 24 days. Additionally, the effects of a combinedtreatment of SNF472 and cilostazol on blood perfusion were also tested.Limb ischemia in the subjects was induced from D1 to D3 excepting thesham group, to which no ischemia was induced. Subjects induced withischemia were then treated with placebo and active agent formulationsfrom D1 to D12 to assess their impact in preventing (a) limb ischemiaand tissue calcification and (b) the deterioration in walking ability.Treatment in all subjects was discontinued from D13 to D24. Observationswere taken at several points during the treatment and post-treatmentphases from D1 to D24. All subjects were weighed every day beforetreatment.

1. Induction of Limb Ischemia

Fifty-four male Sprague Dawley (SD) rats (Envigo Corp., Huntingdon, GB)weighing approximately 250-275 g were used. The subjects were fed withan A04 diet (Scientific Animal Food & Engineering; Carpe Bio,Amersfoort, NL). The subjects were divided in 5 groups, 8 to 10 animalsper group, as follows:

Group 1—Control (sham)

Group 2a Placebo—Physiological saline solution

Group 2b Placebo—5% CMC sodium salt solution

Group 3—SNF472 (Na₆IP₆)

Group 4—Cilostazol (C₂₀H₂₇N₅O₂)

Group 5—Cilostazol+SNF472

Limb ischemia was induced in the subjects of groups 2a-5 by subcutaneousadministration of 120,000 IU/kg vitamin D3 (cholecalciferol, DuphafralD₃ 1000; Zoetis Inc., Parsippany, N.J., US) in physiological salinesolution (2 mL/kg) every day during D1 to D3. The sham group 1 subjectswere administered physiological saline solution (2 mL/kg) subcutaneouslyevery day from D1 to D3 with no vitamin D3.

The subjects in groups 1 and 2a were administered physiological salinesolution (2 mL/kg) subcutaneously every day during D1 to D12. Thesubjects in group 2b were administered a 5% CMC sodium salt in watersolution (5 mL/kg) orally every day during D1 to D12.

2. Ischemia Rescue and Effects on Blood Perfusion—Laser Doppler ImagingAssay

Subjects in groups 1 and 2a were administered 2 mL/kg of physiologicalsaline solution daily from D1 to D12 via subcutaneous route. Subjects ingroup 2b were administered orally 5 mL/kg of a 5% CMC sodium salt inwater solution daily from D1 to D12. In addition, subjects in group 3were administered 20 mg/kg of SNF472 (Na₆IP₆, free base: 600 g/mol)sodium salt (free base 696.27 g/mol) in physiological saline solution (2mL/kg) every day from D1 to D12 via subcutaneous route. Subjects ingroup 4 were administered orally 20 mg/kg cilostazol (C₂₀H₂₇N₅O₂, freebase 369.46 g/mol; lot no. LRAB9590, Sigma-Aldrich Corp., St. Louis,Mo., US) in a 5% CMC sodium salt in water solution (5 mL/kg) daily fromD1 to D12. Subjects in group 5 were administered (i) 20 mg/kg cilostazol(C₂₀H₂₇N₅O₂, free base 369.46 g/mol; lot no. LRAB9590, Sigma-AldrichCorp., St. Louis, Mo., US) in a 5% CMC sodium salt in water solution (5mL/kg) orally, followed by (ii) 20 mg/kg of SNF472 (Na₆IP₆, free base:600 g/mol) in physiological saline solution (2 mL/kg) via subcutaneousroute. The administrations were performed daily from D1 to D12.

On the days when blood perfusion tests involving the use of activeagents were conducted (i.e., D6, D12, and D18), the dosages wereadministered as follows:

Group 3—SNF472 (Na₆IP₆): 15 minutes before reading

Group 4—Cilostazol (C₂₀H₂₇N₅O₂): 3 to 4 hours before reading

Group 5—Cilostazol+SNF472: 15 minutes for SNF472 and 3 to 4 hours forcilostazol before reading

Under the scheme above the active agents would be at their maximumplasma concentration (C_(max)) at the time when the test readings weretaken.

Limb ischemia status was evaluated during treatment and after treatmentwas interrupted (i.e., D0, D6, D12, D18) in all rats by laser dopplerperfusion imaging. The perfusion difference and perfusion ratio werecalculated by comparing the baseline and either of the D6, D12, and D18readings for each group. In particular, the perfusion difference andperfusion ratio were calculated by comparing the group 1 (control) and(a) groups 2a and 2b placebo (physiological saline solution, 5% CMCsodium salt solution), (b) group 3 (SNF472), (c) group 4 (cilostazol),and (d) group 5 (cilostazol/SNF472 combination) readings.

SNF472 by itself or in combination with cilostazol attenuated rat limbischemia. Treatment with cilostazol alone was not effective in thismodel. The SNF472 effects on blood perfusion were maintained even 6 daysafter interrupting treatment. See FIG. 11.

3. Effects on Walking Ability—Treadmill Running Test

Maximal waling time (MWT) and maximal walking distance (MWD) wereassessed in groups 1-4 by the treadmill running test (8 to 10 animalsper group and time point) in D0, D5, D10, and D17.

Subjects were acclimatized to the treadmill for two days beforeconducting the test. On the first day, subjects were exercised for 5 to10 minutes with the treadmill speed ranging progressively from 15 m/minto 24 m/min. On the second day, the subjects were exercised initially ata speed of 15 m/min for the 5 minutes. Then, they were exercised at 19.8m/min (33 cm/sec) for 5 additional minutes. Finally, the subjects wereexercised at 24 m/min (40 cm/sec) for a maximum 30 additional minutes.Preoperative walking time and distance recordings were obtained. Animalsthat did not comply with the protocol were excluded from the test.

Limb function (MWT and MWD) was assessed in groups 1, 2, and 3 using thetreadmill running test 15 min after the corresponding dailyadministration. Limb function in groups 4 was assessed from 3 to 4 hoursafter treatment.

Under the scheme above the active agents would be at their maximumplasma concentration (C_(max)) at the time when the running testreadings were taken.

The subjects were kept running for 40 minutes or until exhausted (i.e.,they remain on the shock grid for five continuous seconds). MWD and MWTwas then calculated for each animal.

SNF472 and cilostazol improved walking ability in rats compared tovehicle (+54% MWD and +46% MWT). See FIG. 13. Moreover, the effect ofSNF472 on improving walking ability was maintained even 5 days afterinterrupting treatment (D17). Contrarily, cilostazol lost its beneficialeffects immediately after treatment was discontinued. See FIG. 13.

4. Calcium Content and Calcification—ICP-OES

Subjects were anesthetized and their blood was obtained in D24. Thesubjects were sacrificed after exsanguination. Then, their necropsieswere performed, and their aortas were collected. The tissues werelyophilized for 24 h and weighed. The lyophilized tissues were thendigested using a 1:1 HNO₃:HClO₄ mixture in a dry bath incubator for 2-4h at 180° C. The digested tissues were subsequently diluted usingultra-pure Milli-Q water (MilliporeSigma (Merck KGaA), Burlington,Mass., US) to a final volume of 10 mL. The calcium content in the tissuesamples was quantified via ICP-OES.

SNF472 showed to be effective against vascular calcification, as SNF472inhibited aorta calcification (41±9%) after daily subcutaneous dosing at20 mg/kg compared to placebo. Cilostazol was not active againstcalcification at the same dose. See FIG. 14.

Example 3 Treatment of Limb Ischemia

The effects of SNF472 and cilostazol over blood perfusion, walkingability and tissue calcification after the inception of limb ischemiawere tested using a rat model for a duration of 13 days. Limb ischemiawas induced to all groups from D1 to D3 excepting the sham group, towhich no ischemia was induced. Subjects induced with ischemia wereadministered placebo and active agent formulations from D5 on to allowfor the development of ischemia before starting treatment. From D5 toD13, subjects were treated from for assessing the impact of treatment onlimb ischemia, walking ability and tissue calcification. Observationswere taken at several points during the treatment from D1 to D13. Allsubjects were weighed every day before treatment.

1. Induction of Limb Ischemia

Sixty-six male Sprague Dawley (SD) rats (Envigo Corp., Huntingdon, GB)weighing approximately 250-275 g were used. The subjects were fed withan A04 diet (Scientific Animal Food & Engineering; Carpe Bio,Amersfoort, NL). The subjects were divided in 5 groups, 8 to 14 animalsper group, as follows:

Group 1—Control (Sham)

Group 2—D5 Ca baseline

Group 3a Placebo—Physiological saline solution

Group 3b Placebo—5% CMC sodium salt solution

Group 4—SNF472 (Na₆IP₆)

Group 5—Cilostazol (C₂₀H₂₇N₅O₂)

Limb ischemia was induced in the subjects of groups 2-5 by subcutaneousadministration of 120,000 IU/kg vitamin D3 (cholecalciferol, DuphafralD₃ 1000; Zoetis Inc., Parsippany, N.J., US) in physiological salinesolution (2 mL/kg) every day during D1 to D3. The group 1 subjects wereadministered physiological saline solution (2 mL/kg) subcutaneouslyevery day from D1 to D3 with no vitamin D3.

2. Ischemia Rescue and Effects on Blood Perfusion—Laser Doppler ImagingAssay

Subjects in group 1 were administered 2 mL/kg of physiological salinesolution daily from D1 to D13 via subcutaneous route. Subjects in group2 were administered 2 mL/kg of physiological saline solution daily fromD1 to D5 via subcutaneous route. On D5, four subjects of group 1 and allthe subjects of group 2 were sacrificed for determining their Cabaseline values.

Subjects in groups 3a placebo were administered 2 mL/kg of physiologicalsaline solution daily from D5 to D13 via subcutaneous route. Subjects ingroup 3b placebo were administered orally 5 mL/kg of a 5% CMC sodiumsalt in water solution daily from D5 to D13. In addition, subjects ingroups 4 were administered 40 mg/kg of SNF472 (Na₆IP₆, free base: 600g/mol) in physiological saline solution (2 mL/kg), every day from D5 toD13 via subcutaneous route.

Subjects in group 5 were administered orally 40 mg/kg cilostazol(C₂₀H₂₇N₅O₂, free base 369.46 g/mol; lot no. LRAB9590, Sigma-AldrichCorp., St. Louis, Mo., US) in a 5% CMC sodium salt in water solution (5mL/kg) daily from D5 to D13. A 40 mg/kg dose of cilostazol in rats iscomparable to a therapeutic dose of 8.4 mg/kg in PAD patients.

On the days when blood perfusion tests involving the use of activeagents were conducted (i.e., D5, D13), the dosages were administered asfollows:

Group 4—SNF472 (Na₆IP₆): 15 minutes before reading

Group 5—Cilostazol (C₂₀H₂₇N₅O₂): 3 to 4 hours before reading

Under the scheme above the active agents would be at their maximumplasma concentration (C_(max)) at the time when the test readings weretaken.

Limb functional and ischemia status were evaluated by laser dopplerperfusion imaging in all groups at D0 and D5, and in groups 1, 3a, 3b, 4and 5 at D13. The perfusion difference and perfusion ratio werecalculated by comparing the baseline and either D5 and D13 readings foreach group. In particular, the perfusion difference and perfusion ratiowere calculated by comparing the group 1 (control) and: (a) the groups3a and 3b placebo (physiological saline solution, 5% CMC sodium saltsolution), (b) the group 4 (SNF472), and (c) the group 5 (cilostazol)readings.

VitD3 administration induced a drop in blood perfusion in the posteriorlimbs in groups 3a, 3b, 4, and 5 (D5 measurement just before therapyadministration). In D13, only animals treated with SNF472 showed asignificant improvement of limbs blood perfusion compared to D5 beforetreatment. No improvement or ischemia rescue were reported in animalstreated with placebo or cilostazol in D13 compared to D5. See FIG. 16.

3. Effects on Walking Ability—Treadmill Running Test

Maximal walking time (MWT) and maximal walking distance (MWD) wereassessed in all groups at D0 and in groups 1, 3a, 3b, 4, and 5 at D6 andD11 by the treadmill running test (8 to 12 animals per group and timepoint). Subjects were acclimatized to the treadmill for two days beforeconducting the test. On the first day, subjects were exercised for 5 to10 minutes with the treadmill speed ranging progressively from 15 m/minto 24 m/min. On the second day, the subjects were exercised initially ata speed of 15 m/min for the 5 minutes. Then, they were exercised at 19.8m/min (33 cm/sec) for 5 additional minutes. Finally, the subjects wereexercised at 24 m/min (40 cm/sec) for a maximum 30 additional minutes.Preoperative walking time and distance recordings were obtained. Animalsthat did not comply with the protocol were excluded from the test.

Limb function (MWT and MWD) was assessed in groups 1, 3a, 3b, and 4using the treadmill running test 15 min after the corresponding dailyadministration. Limb function in groups 5 was assessed from 3 to 4 hoursafter treatment.

Under the scheme above the active agents would be at their maximumplasma concentration (C_(max)) at the time when the test readings weretaken.

The subjects were kept running for 40 minutes or until exhausted (i.e.,they remain on the shock grid for five continuous seconds). MWD and MWTwas then calculated for each animal.

SNF472 improved rat walking ability compared to vehicle (+49% MWD) eventhe treatment started 5 days after ischemia induction whereas cilostazolis not effective under the same conditions and at the therapeutic dose(40 mg/kg/day). See FIG. 16.

4. Calcium Content and Calcification—ICP-OES

Four subjects of group 1 and all the subjects of group 2 were sacrificedon D5 for determining their Ca baseline values. The remaining subjectsof group 1 and all the subjects of groups 3a, 3b, 4, 5, and 6 weresacrificed in D13.

The subjects were sacrificed after exsanguination. Then, theirnecropsies were performed, and their right and left femoral arterieswere collected. The tissues were lyophilized for 24 h and weighed. Thelyophilized tissues were then digested using a 1:1 HNO₃:HClO₄ mixture ina dry bath incubator for 2-4 h at 180° C. The digested tissues weresubsequently diluted using ultra-pure Milli-Q water (MilliporeSigma(Merck KGaA), Burlington, Mass., US) to a final volume of 10 mL. Thecalcium content in the tissue samples was quantified via ICP-OES.

SNF472 showed to be effective against vascular calcification at D13.Compared to placebo, SNF472 inhibited calcification in femoral arteriesby around (30%) after a daily subcutaneous dosing of 40 mg/kg. See FIG.17.

5. Pharmacokinetics

Subjects from groups, 1, 3a,3b, 4, and 5 were anesthetized and theirblood was obtained in D13. Around 8-10 mL of total blood per subjectwere taken and divided in collection tubes for plasma (K3EDTA,approximately 6 mL blood) and serum (approximately 2-3 mL blood)testing. Plasma was be stored in one aliquot of 600 μL and several otheraliquots of 500 μL. Serum was be divided in two aliquots.

Myo-inositol-hexaphosphate levels in the plasma of the subjects of group4 (i.e., taken at around C_(max), 15 min after the last SNF472 dosing)were quantified by LC-MS/MS chromatographic methods described in theart. See WO2013050603.

All subjects in group 4 were well exposed to the SNF472 product. Theplasma level 15 min after the last subcutaneous dosing in D13 was40078±15024 ng/mL (60.7±22.8 uM). The plasma levels in rats after adaily dosing at 20 mg/kg were comparable to the levels found inhemodialysis patients treated with SNF472 at a dose of 8.4 mg/kg via theintravenous route.

Example 4 SN472 Drug Interactions

The compatibility of SNF472 with other drugs prescribed regularly tosubjects with renal impairment was analyzed.

Wistar rats (Charles River Labs, Inc., Wilmington, Mass., US), weretreated with: (i) SNF472 administered subcutaneously (s.c.), (ii)SNF472+sevelamer (oral), (iii) SNF472 (s.c.)+cinacalcet (oral), (iv)SNF472(s.c.)+Vit D (s.c.), (v) SNF472 (s.c.)+sodium thiosulfate (s.c.),and SNF472 (s.c.)+ibandronate (s.c.). No significant differences wereobserved between the administration of SNF472 alone or jointly with anyother of the assayed drugs.

Example 5 Prevention of Limb Ischemia SNF472 Dose-Response Impact OverMaximal Walking (MWT) and Distance (MDT) Times

The preventive effects of several different doses of SNF472 and thehighest tolerated cilostazol dose on blood perfusion, walking abilityand tissue calcification were tested for 12 days using a rat model. Limbischemia was induced in the subjects from D1 to D3, excepting the shamgroup. Animals induced with ischemia were then treated with placebo andactive agent formulations from D1 to D12 to assess their impact inpreventing (a) limb ischemia and tissue calcification and (b) thedeterioration in walking ability. Observations were taken at severalpoints during the treatment phase from D1 to D12. All subjects wereweighed every day before treatment.

1. Induction of Limb Ischemia

One hundred and two male Sprague Dawley (SD) rats (Envigo Corp.,Huntingdon, GB) weighing approximately 250-275 g were used. The subjectswere fed with an A04 diet (Scientific Animal Food & Engineering; CarpeBio, Amersfoort, NL). The subjects were divided in 5 groups, 8 to 12animals per group, as follows:

Group 1—Control (sham)

Group 2a Placebo—Physiological saline solution, subcutaneous

Group 2b Placebo—5% CMC sodium salt solution, oral

Group 3—SNF472 (Na₆IP₆) at 1 mg/kg, subcutaneous

Group 4—SNF472 (Na₆IP₆) at 7.5 mg/kg, subcutaneous

Group 5—SNF472 (Na₆IP₆) at 15 mg/kg, subcutaneous

Group 6—SNF472 (Na₆IP₆) at 30 mg/kg, subcutaneous

Group 7—SNF472 (Na₆IP₆) at 45 mg/kg, subcutaneous

Group 8—Cilostazol (C₂₀H₂₇N₅O₂) at 45 mg/kg, oral

Limb ischemia was induced in the subjects of groups 2-8 by subcutaneousadministration of 120,000 IU/kg vitamin D3 (cholecalciferol, DuphafralD₃ 1000; Zoetis Inc., Parsippany, N.J., US) in physiological salinesolution (2 mL/kg) every day during D1 to D3. The sham group 1 subjectswere administered physiological saline solution (2 mL/kg) subcutaneouslyevery day from D1 to D3 with no vitamin D3.

The subjects in groups 1 and 2a were administered physiological salinesolution (2 mL/kg) subcutaneously every day during D1 to D12. Thesubjects in group 2b were administered a 5% CMC sodium salt in watersolution (5 mL/kg) orally every day during D1 to D12.

2. Ischemia Rescue and Effects on Blood Perfusion—Laser Doppler ImagingAssay

Subjects in groups 1 and 2a were administered 2 mL/kg of physiologicalsaline solution daily from D1 to D12 via subcutaneous route. Subjects ingroup 2b were administered orally 5 mL/kg of a 5% CMC sodium salt inwater solution every day from D1 to D12. In addition, subjects in group3, 4, 5, 6 and 7 were administered 1 mg/kg, 7.5 mg/kg, 15 mg/kg, 30mg/kg and 45 mg/kg of SNF472 (Na₆IP₆, free base: 600 g/mol) sodium salt(free base 696.27 g/mol), respectively. SNF472 was administrated inphysiological saline solution (2 mL/kg) every day from D1 to D12 viasubcutaneous route. Subjects in group 8 were administered orally 45mg/kg cilostazol (C₂₀H₂₇N₅O₂, free base 369.46 g/mol; lot no. LRAB9590,Sigma-Aldrich Corp., St. Louis, Mo., US) in a 5% CMC sodium salt inwater solution (5 mL/kg) daily from D1 to D12.

On the days when blood perfusion tests involving the use of activeagents were conducted (i.e., D6 and D12), the dosages were administeredas follows:

Groups 3, 4, 5, 6 and 7—SNF472 (Na₆IP₆): 15 minutes before reading

Group 8—Cilostazol (C₂₀H₂₇N₅O₂): 3 to 4 hours before reading

Under the scheme above the active agents would be at their maximumplasma concentration (C_(max)) at the time when the test readings weretaken.

Limb ischemia status was evaluated during the treatment period (i.e.,D0, D6 and D12) by laser doppler perfusion imaging in all rats. Theperfusion difference and perfusion ratio were calculated by comparingthe baseline and either of the D6 and D12 readings for each group. Inparticular, the perfusion difference and perfusion ratio were calculatedby comparing the group 1 (control) and (a) groups 2a and 2b placebo(physiological saline solution, 5% CMC sodium salt solution), (b) groups3, 4, 5, 6 and 7 (SNF472) and (c) group 8 (cilostazol), readings.

SNF472 attenuated rat limb ischemia with dose-response manner. Treatmentwith cilostazol alone was not effective in this model.

3. Effects on Walking Ability—Treadmill Running Test

Maximal walking time (MWT) and maximal walking distance (MWD) wereassessed in groups 1-8 by the treadmill running test (8 to 12 animalsper group and time point) in D0, D5 and D10.

Subjects were acclimatized to the treadmill for two days beforeconducting the test. On the first day, subjects were exercised for 5 to10 minutes with the treadmill speed ranging progressively from 15 m/minto 24 m/min. On the second day, the subjects were exercised initially ata speed of 15 m/min for the 5 minutes. Then, they were exercised at 19.8m/min (33 cm/sec) for 5 additional minutes. Finally, the subjects wereexercised at 24 m/min (40 cm/sec) for a maximum 30 additional minutes.Preoperative walking time and distance recordings were obtained. Animalsthat did not comply with the protocol were excluded from the test.

Limb function (MWT and MWD) was assessed in groups 1, 2, 3, 4, 5, 6 and7 using the treadmill running test 15 min after the corresponding dailyadministration. Limb function in group 8 was assessed from 3 to 4 hoursafter treatment.

Under the scheme above the active agents would be at their maximumplasma concentration (C_(max)) at the time when the running testreadings were taken.

The subjects were kept running for 40 minutes or until exhausted (i.e.,they remain on the shock grid for five continuous seconds). MWD and MWTwas then calculated for each animal.

SNF472 and cilostazol improved walking ability in rats compared tovehicle. Moreover, the effect of SNF472 on improving walking ability wasdose-response dependent.

4. Calcium Content and Calcification—ICP-OES

Subjects were anesthetized and their blood was obtained in D12. Thesubjects were sacrificed after exsanguination. Then, their necropsieswere performed, and their heart and aorta arteries were collected. Thetissues were lyophilized for 24 h and weighed. The lyophilized tissueswere then digested using a 1:1 HNO₃:HClO₄ mixture in a dry bathincubator for 2-4 h at 180° C. The digested tissues were subsequentlydiluted using ultra-pure Milli-Q water (MilliporeSigma (Merck KGaA),Burlington, Mass., US) to a final volume of 10 mL. The calcium contentin the tissue samples was quantified via ICP-OES.

SNF472 showed to be effective against heart and vascular calcification,such as, for example, heart and aorta arteries calcification, in adose-response manner. Cilostazol was not active against calcification atthe same dose.

Example 6 Prevention of Limb Ischemia in an Adenine-Induced Uremic RatModel SNF472 Impact on Limb Blood Perfusion

The preventive effects of SNF472 and cilostazol doses on blood perfusionand tissue calcification were tested using a relevant chronic kidneydisease rat model for a duration of 21 days.

Uremia and limb ischemia in the animals were induced from D1 to D21excepting the sham group, to which neither uremia nor ischemia wasinduced. Animals induced with ischemia were then treated with placeboand active agent formulations from D1 to D21 to assess their impact inpreventing (a) limb ischemia and (b) tissue calcification. Observationswere taken at several points during the treatment phase from D1 to D21.All subjects were weighed every day before treatment.

1. Induction of Uremia and Limb Ischemia

Sixty-eight male Sprague Dawley (SD) rats (Envigo Corp., Huntingdon, GB)weighing approximately 250-275 g were used. fed a pelletedhigh-phosphorus diet (SM R, 10 mm pellets, 1.06% Ca, 1.03% P) (SSNIFFSpezialdiaeten, Soest, DE) ad libitum. The subjects were divided in 6groups, 8 to 12 animals per group, as follows:

Group 1—Control (sham)

Group 2a Placebo—Physiological saline solution subcutaneous, once a day

Group 2b Placebo—Physiological saline solution, subcutaneous, Alzet pump4 weeks

Group 3—SNF472 (Na₆IP₆) at 30 mg/kg, subcutaneous, once a day

Group 4—SNF472 (Na₆IP₆) at 45 mg/kg, subcutaneous, once a day

Group 5—SNF472 (Na₆IP₆) at total dose of 400 mg/4 weeks (100 mg/week),subcutaneous, Alzet pump 4 weeks was implanted in D1 before adenineadministration

Group 6—Cilostazol (C₂₀H₂₇N₅O₂) at 45 mg/kg/day, oral, once a day

Uremia and limb ischemia were induced in groups 2-6 by a daily dose ofadenine (500 mg/kg, suspended in 1% carboxymethyl cellulose,administered orally) for the first 10 days, and then with a dose ofα-calcidol (100 ng/kg in olive oil, administered orally) three times perweek from D11 to D19, to accelerate and homogenize the development ofcardiovascular calcification and ischemia.

On day 21, animals were sacrificed, and blood and tissue samples werecollected. Creatinine (Ref no. OSR6178) and urea (Ref. no. OSR6134)levels in serum were determined using the corresponding Beckman Coulterassay kit (Beckman Coulter, Inc., Brea, Calif., US).

The sham group 1 animals were administered 1% carboxymethyl cellulosesolution (5 mL/kg) orally every day from D1 to D10 and then wereadministered orally olive oil three times per week from D11 to D19.Neither uremia nor ischemia were induced in sham group.

2. Ischemia Rescue and Effects on Blood Perfusion—Laser Doppler ImagingAssay

The subjects in groups 2a were administered physiological salinesolution (2 mL/kg) subcutaneously twice a day during 21 days. Thesubjects in group 2b were administered physiological saline solution viasubcutaneous 4 weeks using an Alzet pump.

In addition, animals in group 3 and 4 were administered subcutaneouslytwice a day at 30 mg/kg and 45 mg/kg of SNF472 (Na₆IP₆, free base: 600g/mol) sodium salt (free base 696.27 g/mol), respectively. SNF472 wasadministrated in physiological saline solution (2 mL/kg) twice a dayfrom D1 to D21 via subcutaneous route. The animals in group 5 wereadministered SNF472 at 400 mg/4 weeks dissolved physiological salinesolution via subcutaneous 4 weeks Alzet pump.

Animals in group 6 were administered orally 45 mg/kg cilostazol(C₂₀H₂₇N₅O₂, free base 369.46 g/mol; lot no. LRAB9590, Sigma-AldrichCorp., St. Louis, Mo., US) in a 5% CMC sodium salt in water solution (5mL/kg) daily from D1 to D21.

On the days when blood perfusion tests involving the use of activeagents were conducted (i.e., D10, D17, and D21), the dosages wereadministered as follows:

Groups 3 and 4—SNF472 (Na₆IP₆): 15 minutes before reading

Group 6—Cilostazol (C₂₀H₂₇N₅O₂): 3 to 4 hours before reading

Under the scheme above the active agents would be at their maximumplasma concentration (C_(max)) at the time when the test readings weretaken.

Limb ischemia status was evaluated during treatment (i.e., D0, D10 andD17 and D21) in all rats by laser doppler perfusion imaging. Theperfusion difference and perfusion ratio were calculated by comparingthe baseline and of the D10, D17 and D21 readings for each group. Inparticular, the perfusion difference and perfusion ratio were calculatedby comparing the group 1 (control) and (a) groups 2a and 2b placebo, (b)groups 3, 4 and 5 (SNF472) and (c) group 6 (cilostazol), readings.

SNF472 attenuated rat limb ischemia in uremic rats in a dose-responsemanner. Treatment with cilostazol alone was not effective in this model.

3. Calcium Content and Calcification—ICP-OES

Subjects were anesthetized and their blood was obtained in D21. Thesubjects were sacrificed after exsanguination. Then, their necropsieswere performed, and their heart and aorta arteries were collected. Thetissues were lyophilized for 24 h and weighed. The lyophilized tissueswere then digested using a 1:1 HNO₃:HClO₄ mixture in a dry bathincubator for 2-4 h at 180° C. The digested tissues were subsequentlydiluted using ultra-pure Milli-Q water (MilliporeSigma (Merck KGaA),Burlington, Mass., US) to a final volume of 10 mL. The calcium contentin the tissue samples was quantified via ICP-OES.

SNF472 showed to be effective against heart and vascular calcificationin uremic rats, such as, for example, in heart and aorta arteriescalcification, in a dose-response manner after daily subcutaneous dosingor/and delivered by Alzet pump compared to placebo. Cilostazol was notactive against calcification at the same dose.

1. A method of increasing tissular perfusion and/or oxygenation in asubject in need thereof comprising administering a compound of generalformula I, or a pharmaceutically acceptable salt thereof to the subject

wherein R₁, R₃, R₅, R₇, R₉ and R₁₁ are independently selected from OH, aradical of formula II, III, IV and a heterologous moiety:

and wherein: (i) at least one of R₁, R₃, R₅, R₇, R₉ and R₁₁ is selectedfrom a radical of formula II, III and IV, and (ii) zero, one, two orthree of R₁, R₃, R₅, R₇, R₉ and R₁₁ is a heterologous moiety. 2.(canceled)
 3. The method of claim 1, wherein the heterologous moiety isselected from a radical of formula V, a radical of formula VI and aradical of formula VII:

wherein: n is an integer in the range from 2 to 200, and R₁₃ is selectedfrom H, methyl and ethyl.
 4. The method of claim 1, wherein the subjectsuffers from peripheral arterial disease or is at risk of developingperipheral arterial disease.
 5. The method of claim 1, wherein thesubject suffers from critical limb ischemia or is at risk of developingcritical limb ischemia.
 6. The method of claim 1, wherein thepharmaceutically acceptable salt is a sodium salt.
 7. The method ofclaim 1, wherein the compound of formula I is inositol hexaphosphate. 8.The method of claim 7, wherein the inositol hexaphosphate ismyo-inositol hexaphosphate.
 9. The method of claim 7, wherein theinositol hexaphosphate is a hexasodium salt.
 10. The method of claim 1,wherein one or two of R₁, R₃, R₅, R₇, R₉ and R₁₁ is selected from aradical of formula V, VI and VII.
 11. The method of claim 10, whereinR₁, R₅, R₉ and R₁₁ are a radical of formula II and R₃ and R₇ are aradical of formula V.
 12. The method of claim 11, wherein the radical offormula V has n in the range from 2 to 200 and R₁₃ is H.
 13. The methodof claim 1, wherein the compound or pharmaceutically acceptable saltthereof is in a pharmaceutical composition comprising pharmaceuticallyacceptable excipients and carriers.
 14. The method of claim 1, whereinthe subject is a subject with kidney failure.
 15. The method of claim 1,wherein the compound or pharmaceutically acceptable salt thereof isadministered to the subject during dialysis.
 16. The method of claim 1,wherein the compound or pharmaceutically acceptable salt thereof isadministered to the subject during hemodialysis.
 17. The method of claim1, wherein the compound or pharmaceutically acceptable salt thereof isadministered to an unfiltered blood extracted from the subject.
 18. Themethod of claim 1, wherein the compound or pharmaceutically acceptablesalt thereof is administered by parenteral route.
 19. The method ofclaim 18, wherein the parenteral route is intravenous, subcutaneous orintramuscular.
 20. (canceled)
 21. (canceled)
 22. The method of claim 1,wherein the compound or pharmaceutically acceptable salt thereof isadministered to the subject in an effective dosage of about 0.001 mg/kgto about 60 mg/kg.
 23. A method of treating or preventing ischemiaand/or an ischemia-related disease or condition in a subject in needthereof, comprising administering a compound of general formula I, or apharmaceutically acceptable salt thereof to the subject

wherein R₁, R₃, R₅, R₇, R₉ and R₁₁ are independently selected from OH, aradical of formula II, III, IV and a heterologous moiety:

wherein: (i) at least one of R₁, R₃, R₅, R₇, R₉ and R₁₁ is selected froma radical of formula II, III and IV, and (ii) zero, one, two or three ofR₁, R₃, R₅, R₇, R₉ and R₁₁ is a heterologous moiety, and wherein thesubject suffers from peripheral arterial disease or is at risk ofdeveloping peripheral arterial disease, the subject suffers fromcritical limb ischemia or is at risk of developing critical limbischemia, the subject has kidney failure or is at risk of sufferingkidney failure, or the subject is undergoing hemodialysis.
 24. Themethod of claim 23, wherein the ischemia is limb ischemia.
 25. A methodof improving the walking ability of a subject in need thereof,comprising administering a compound of general formula I, or apharmaceutically acceptable salt thereof to the subject

wherein R₁, R₃, R₅, R₇, R₉ and R₁₁ are independently selected from OH, aradical of formula II, III, IV and a heterologous moiety:

wherein: (i) at least one of R₁, R₃, R₅, R₇, R₉ and R₁₁ is selected froma radical of formula II, III and IV, and (ii) zero, one, two or three ofR₁, R₃, R₅, R₇, R₉ and R₁₁ is a heterologous moiety, and wherein thesubject suffers from peripheral arterial disease or is at risk ofdeveloping peripheral arterial disease, the subject suffers fromcritical limb ischemia or is at risk of developing critical limbischemia, the subject has kidney failure or is at risk of sufferingkidney failure, or the subject is undergoing hemodialysis.
 26. Themethod of claim 25, wherein the increase in limb function improves thewalking ability of the subject.
 27. The method of claim 25, wherein theincrease in limb function is determined by Maximal Walking Distance(MWD), Maximal Walking Time (MWT) or both.
 28. The method of claim 1,wherein the compound is SNF472.
 29. The method of claim 1, wherein thecompound is administered as a dose between 2.5 mg/kg and 5 mg/kg,between 5 mg/kg and 7.5 mg/kg, between 7.5 mg/kg and 10 mg/kg, orbetween 5 mg/kg and 10 mg/kg.
 30. The method of claim 1, wherein thecompound is administered at 300 mg per dose.
 31. A method to treat orinhibit the development of peripheral arterial disease or critical limbischemia in a subject comprising administering to the subject atherapeutically effective dose of inositol hexaphosphate between 5 mg/kgand 10 mg/kg.
 32. The method of claim 31, wherein the dose of inositolhexaphosphate is between 7.5 mg/kg and 10 mg/kg.
 33. The method of claim31, wherein the dose of inositol hexaphosphate is administeredintravenously or subcutaneously.